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

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 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 10ng/ml TGFβ2; Days 4-7, 2% Human serum and 1ng/ml TGFβ2; Days 8-14, 1% Human serum and 0.1ng/ml TGFβ2; 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 trans-differentiation 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 trans-differentiation 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 post-surgical wound-healing and PCO than the Alcon Acrysof using this model

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 β (TGFβ2); days 4–7, 2% human serum and 1 ng/mL TGFβ2; days 8–14, 1% human serum and 0.1 ng/mL TGFβ2; 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

The MH1 domain of Smad3 interacts with Pax6 and represses autoregulation of the Pax6 P1 promoter

Pax6 transcription is under the control of two main promoters (P0 and P1), and these are autoregulated by Pax6. Additionally, Pax6 expression is under the control of the TGFβ superfamily, although the precise mechanisms of such regulation are not understood. The effect of TGFβ on Pax6 expression was studied in the FHL124 lens epithelial cell line and was found to cause up to a 50% reduction in Pax6 mRNA levels within 24 h. Analysis of luciferase reporters showed that Pax6 autoregulation of the P1 promoter, and its induction of a synthetic promoter encoding six paired domain-binding sites, were significantly repressed by both an activated TGFβ receptor and TGFβ ligand stimulation. Subsequently, a novel Pax6 binding site in P1 was shown to be necessary for autoregulation, indicating a direct influence of Pax6 protein on P1. In transfected cells, and endogenously in FHL124 cells, Pax6 co-immunoprecipitated with Smad3 following TGFβ receptor activation, while in GST pull-down experiments, the MH1 domain of Smad3 was observed binding the RED sub-domain of the Pax6 paired domain. Finally, in DNA adsorption assays, activated Smad3 inhibited Pax6 from binding the consensus paired domain recognition sequence. We hypothesize that the Pax6 autoregulatory loop is targeted for repression by the TGFβ/Smad pathway, and conclude that this involves diminished paired domain DNA-binding function resulting from a ligand-dependant interaction between Pax6 and Smad3

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

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

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 10µM 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 ER stress genes, which also corresponded with protein expression. Evidence of autophagy was observed in response to SFN as determined by increased 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

Aspirin Inhibits TGFβ2-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 β2 (TGFβ2) has been considered an obligatory mechanism for PCO. In this study, we tested the efficacy of aspirin in inhibiting the TGFβ2-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 TGFβ2 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 TGFβ2-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 TGFβ2-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 TGFβ2-mediated EMT of LECs, possibly from epigenetic down-regulation of EMT-related genes