The lens as a model for fibrotic disease

Fibrosis affects multiple organs and is associated with hyperproliferation, cell transdifferentiation, matrix modification and contraction. It is therefore essential to discover the key drivers of fibrotic events, which in turn will facilitate the development of appropriate therapeutic strategies. The lens is an elegant experimental model to study the processes that give rise to fibrosis. The molecular and cellular organization of the lens is well defined and consequently modifications associated with fibrosis can be clearly assessed. Moreover, the avascular and non-innervated properties of the lens allow effective in vitro studies to be employed that complement in vivo systems and relate to clinical data. Using the lens as a model for fibrosis has direct relevance to millions affected by lens disorders, but also serves as a valuable experimental tool to understand fibrosis per se

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

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

Regional differences in store-operated Ca2+ entry in the epithelium of the intact human lens

An elevated level of Ca2+ is an important factor in cataract, yet precisely how Ca2+ enters the lens is unknown. Lens epithelial cells contain a range of G-protein–coupled receptors and receptor tyrosine kinases that induce increases in intracellular Ca2+. Receptor-associated Ca2+ influx is, therefore, likely to be an important route for Ca2+ influx to the lens. The authors investigated stimulated and passive Ca2+ influx in in situ human lens epithelium. Ca2+ changes in equatorial (E) and central anterior (CA) epithelial cells were monitored with the use of a Ca2+ indicator (Fluo4) and confocal microscopy. Gene expression was monitored by RT-PCR and immunoblotting. Adenosine triphosphate (ATP) induced Ca2+ responses that were smaller in CA than E. Ca2+ store depletion, using ATP (100 µM) or thapsigargin (1 µM), revealed greater relative store capacity and Ca2+ influx in E. Ca2+ influx was blocked by La3+ (0.5 µM) in both regions. Unstimulated Ca2+ influx was greater in E than CA. Greater expression of Orai1 and STIM1 was detected in E than in CA. Greater Ca2+ store capacity and Ca2+ influx in E compared with CA reflects underlying differences in proliferation and differentiation between the regions. The relatively small resting Ca2+ influx in CA epithelium suggests that store-operated Ca2+ entry (SOCE) is the main route of Ca2+ influx in these cells. Greater resting influx and SOCE in E cells suggests that these are a major route for Ca2+ influx into the lens. Increased expression of Orai1 and STIM1 in E could account for the differences in Ca2+ entry. Receptor activation will modulate Ca2+ influx, and inappropriate activity may contribute to cortical cataract

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

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 employed as experimental systems. Standard culture was in 5% FCS EMEM. 10ng/ml Transforming growth factor-2 (TGFβ2) was used to induce fibrotic changes. A scratch wound assay was used to measure cell migration and the patch assay employed to assess matrix contraction by FHL124 cells. Protein expression was assessed by immunocytochemistry and western blot and gene expression by QRT-PCR. In capsular bags, cell growth across the posterior lens capsule, capsular wrinkling and EMT 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 TGFβ2-induced expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) at both the message and protein level as well as significantly inhibiting matrix contraction induced by TGFβ2. In human capsular bags, 30 μM RESV significantly inhibited cell growth. TGFβ2-induced α-SMA expression and capsular wrinkling were also significantly inhibited by RESV treatment. RESV significantly supressed expression of TGFβ2-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

Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens

PURPOSE: Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens. METHODS: The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software. RESULTS: Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls. CONCLUSIONS: Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation

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