FHL124 Cells Research Articles

Upregulation of histone H3 caused by CRYAA may contribute to the development of age-related cataract

Objective: Age-relate cataract (ARC) is a disease of the eyes with no effective drugs to prevent or treat patients. The aim of the present study is to determine whether histone H3, αA-crystallin (CRYAA), β-galactosidase (GLB1), and p53 are involved in the pathogenesis of ARC. Methods: A total of 99 anterior lens capsules (ALCs) of patients with ARC of various nuclear grades, ultraviolet models of ALCs, and two human lens epithelial cell lines (FHL-124 and SRA01/04) were used, and the expression of histone H3, CRYAA, GLB1, and p53 were detected by immunoblotting and reverse transcription and real time-quantitative polymerase chain reaction. The association between CRYAA with histone H3, GLB1, and p53 was assessed in FHL-124 and SRA01/04 cells following CRYAA overexpression. Results: Histone H3 and p53 in ALCs of patients with ARC were up-regulated in a grade-dependent manner, and the expression of CRYAA showed a positive association with histone H3, p53, and GLB1. In UV models of ALCs and human lens epithelial cell lines, the expression levels of histone H3, cell apoptosis factors (Bax/Bcl-2, cleaved caspase-3), and inflammation factors (interleukin-6, tumor necrosis factor-α) were all up-regulated. Furthermore, transfection of CRYAA in FHL-124 cells induced overexpression of histone H3. Conclusion: CRYAA-mediated upregulation of histone H3 may be involved in the pathogenesis of ARC. p53 may also have a role in ARC development, but not via the CRYAA-histone H3 axis. The results of the present study may assist in improving our understanding of the pathogenesis of ARC and in identifying potential targets for treatment.

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

PurposeSulforaphane (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.MethodsThe 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.ResultsTreatment 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.ConclusionsSFN 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.

Development of a drug-eluting intraocular lens to deliver epidermal growth factor receptor inhibitor gefitinib for posterior capsule opacification prophylaxis.

Different molecular targets, such as the epidermal growth factor receptor, have been identified for the prophylaxis of posterior capsule opacification. This led to the proposal of several drugs, yet drug delivery into the capsular bag remains challenging. The intraocular lens as a drug delivery device would provide a convenient method to allow drug release in the location needed. This is to evaluate the effect of a drug-eluting intraocular lens using an epidermal growth factor receptor inhibitor. Hydrophobic and hydrophilic intraocular lenses were coated with gefitinib using the dip coating technique. The cellular response on the modified intraocular lenses was tested in a human lens epithelial cell line (FHL-124) in an anterior segment model. Furthermore, modified intraocular lenses were implanted into human capsular bags ex vivo. Drug release was determined as well as the biocompatibility on human corneal endothelial cells. Unmodified intraocular lenses served as controls. In addition, immunofluorescence staining with fibronectin as a marker for fibrotic response was conducted. Both coated hydrophilic and hydrophobic intraocular lenses could attenuate the cell growth of FHL-124 cells in the human capsular bag in comparison to the unmodified controls. Furthermore, gefitinib-soaked intraocular lenses showed a constant drug release over the first 10 days. No reduction in cell viability of corneal endothelial cells occurred. A decrease in fibronectin expression under gefitinib treatment could be observed. In vitro epidermal growth factor receptor seems to be a valuable target for the prevention of posterior capsule opacification. The gefitinib-eluting intraocular lens in this study could inhibit cell growth in non-toxic concentrations.

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

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. 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-β2 (TGFβ2) 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. 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 levels, 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 suppressed expression of TGFβ2-induced genes associated with fibrotic disease, including matrix metalloproteinase-2 in FHL124 cells, capsular bags, and central anterior epithelium. 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.

Localization Analysis of Seven De-sumoylation Enzymes (SENPs) in Ocular Cell Lines.

It is now well established that protein sumoylation acts as an important regulatory mechanism modulating functions over three thousand proteins. In the vision system, protein conjugation with SUMO peptides can regulate differentiation of multiple ocular tissues. Such regulation is often explored through analysis of biochemical and physiological changes with various cell lines in vitro. We have recently analyzed the expression levels of both mRNAs and proteins for seven de-sumoylation enzymes (SENPs) in five major ocular cell lines. In continuing the previous study, here we have determined their cellular localization of the seven de-sumoylation enzymes (SENP1, 2, 3, 5, 6, 7 and 8) in the above 5 major ocular cell lines using immunocytochemistry. The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The localization of the 7 major de-sumoylation enzymes (SENPs) in the 5 major ocular cell lines were determined with immunohistochemistry. The images were captured with a Zeiss LSM 880 confocal microscope. 1) The SENP1 was localized in both cytoplasm and nucleus of 3 human ocular cell lines, FHL124, HLE and ARPE-19; In N/N1003A and αTN4-1, SENP 1 was more concentrated in the cytoplasm. SENP1 appears in patches; 2) SENP2 was distributed in both cytoplasm and nucleus of all ocular cell lines in patches. In HLE and ARPE-19 cells, SENP2 level was higher in nucleus than in cytoplasm; 3) SENP3 was almost exclusively concentrated in the nuclei in all ocular cells except for N/N1003A cells. In the later cells, a substantial amount of SENP3 was also detected in the cytoplasm although nuclear SENP3 level was higher than the cytoplasmic SENP3 level. SENP3 appeared in obvious patches in the nuclei; 4) SENP5 was dominantly localized in the cytoplasm (cellular organelles) near nuclear membrane or cytoplasmic membrane ; 5) SENP6 was largely concentrated in the nuclei of all cell lines except for αTN4-1 cells. In the later cells, a substantial amount of SENP6 was also detected in the cytoplasm although nuclear SENP6 level was higher than the cytoplasmic SENP6 level. 6) SENP7 has an opposite localization pattern between human and animal cell lines. In human cell lines, a majority of SENP7 was localized in nuclei whereas in mouse and rabbit lens epithelial cells, most SENP7 was distributed in the cytoplasm. SENP8 was found present in human cell lines. The 3 human ocular cell lines had relatively similar distribution pattern. In FHL124 and ARPE-19 cells, SENP8 was detected only in the cytoplasm, but in HLE cells, patches of SENP8 in small amount was also detected in the nuclei. Our results for the first time defined the differential distribution patterns of seven desumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to understand the different functions of various SENPs in maintaining the homeostasis of protein sumoylation patterns during their functioning processes.

The intraocular lens as a drug delivery device for an epidermal growth factor-Receptor inhibitor for prophylaxis of posterior capsule opacification.

Posterior capsule opacification (PCO) occurs as a common complication after cataract surgery. Erlotinib is an inhibitor of the epidermal growth factor-Receptor and reduces critical cellular events leading to PCO. In this invitro study, Erlotinib-modified intraocular lenses (IOLs) employed as a drug delivery device have been evaluated for PCO prevention. The IC50 concentration of Erlotinib was determined by using FHL-124 cells. For the human capsular bag model, 40 cadaver eyes underwent sham cataract surgery. Sixteen capsular bags were exposed to the IC50 of Erlotinib. Intraocular lens (IOL) of three different materials was pharmacologically modified and tested in the anterior segment model and implanted into 24 capsular bags. To test for corneal toxicity, pairs of human cornea were exposed to high concentrations of Erlotinib and corneal endothelial cells (CEC) were exposed to the modified IOL. Release kinetics of Erlotinib from the IOL was measured. IC50 of Erlotinib was determined to be 10μm. Erlotinib alone (p=0.002) and when soaked into IOLs (p<0.001) significantly increased the number of days needed until total cell coverage of the capsular bags in comparison with the control. Modified IOLs mitigated cell growth in the anterior segment model (p<0.001). No short-term corneal toxicity was observed up to a concentration of 100μm, and IOLs did not show toxicity on CEC. Erlotinib was released constantly from IOL. Erlotinib might be of clinical relevance in PCO prophylaxis, as its short-term application induces a long-term deceleration of cellular growth. Erlotinib can be introduced into the eye via soaked IOLs.

The role of HIF-1α in the TGF-β2-mediated epithelial-to-mesenchymal transition of human lens epithelial cells.

Human lens epithelial cells (HLE) undergo mesenchymal transition and become fibrotic during posterior capsule opacification (PCO), which is a frequent complication after cataract surgery. TGF-β2 has been implicated in this fibrosis. Previous studies have focused on the role of hypoxia-inducible factor-1α (HIF-1α) in fibrotic diseases, but the role of HIF-1α in the TGF-β2-mediated fibrosis in HLE is not known. TGF-β2 treatment (10 ng/mL, 48 h) increased the HIF-1α levels along with the EMT markers in cultured human lens epithelial cells (FHL124 cells). The increase in HIF-1α corresponded to an increase in VEGF-A in the culture medium. However, exogenous addition of VEGF-A (up to 10 ng/mL) did not alter the EMT marker levels in HLE. Addition of a prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, up to 10 µM), enhanced the levels of HIF-1α, and secreted VEGF-A but did not alter the EMT marker levels. However, treatment of cells with a HIF-1α translational inhibitor, KC7F2, significantly reduced the TGF-β2-mediated EMT response. This was accompanied by a reduction in the ERK phosphorylation and nuclear translocation of Snail and Slug. Together, these data suggest that HIF-1α is important for the TGF-β2-mediated EMT of human lens epithelial cells.

Cytosolic Internalization of the Na/K ATPase Ouabain Receptor Complex (NORC)

In early (1960–80) classic work, digitalis (ouabain) binding to the Na/K ATPase (NKA) was considered mainly a disruption of the cellular ionic steady state and in terms of ligand dependency. During the past 2–3 decades, the role of NKA as signal transducer involving protein/protein interactions with subsequent protein phosphorylation and gene transcriptional upregulation was elucidated regarding cell survival and apoptosis. Recently re‐established, the significance of a cytosolic internalization of the NKA‐ouabain receptor‐complex (here called NORC) is not yet understood in terms of details of participating proteins and organelles, and its potential for gene transcriptional upregulation. The fate of NORC was followed by immunocytochemistry, and enhanced dark field hyperspectral microscopy (EDHM) in two different lens epithelial cells (LECs) (FHL124 and B3) with the aim to characterize some of the protein partners of the hypothetical membrane‐to‐cytosol escalator involved in this process. Partners of interest were the Bcl‐2 protein BclXL, proposed by us to interact with NKA's N‐terminal domain (Lauf et al., Cell Physiol Biochem 31:257–276, 2013) and detected by co‐immunoprecipitation (Lauf et al., Am J Physiol Cell Physiol. 308, C51–60, 2015), α‐tubulin (Zampar et al. Biochem J. 422:129,2009), caveolin (Cui &amp; Zie, Molecules 22:990, 2017), and the endosomal and lysosomal proteins EEA1 and LAMP1 (Cheriavsky et al., JBC 289:1049, 2014). The presence of these protein partners was also studied in B3 LECs and verified by PAGEL and Western blots. Protein interactions were tested with Bodipy‐green fluorescent ouabain (BFO) and green‐ or red‐fluorescent labeled antibodies against all protein components, verified immuno‐cytochemically by overlapping yellow/orange fluorescence and further refined by MATLAB photo‐editing for background noise. Results: Based on immuno‐colocalization of BFO with Cy‐3 Red‐stained anti‐α1 NKA isoform antibody, NORC transgressed the plasmalemma into cytosol 45 min after exposure to μM BFO. This was further supported by finding of intracellular BFO labeling using EDHM in FHL124 cells. Higher ouabain concentrations affected cell size and NKA distribution. However, interaction of NKA with Bcl‐XS, a shorter variant of BclXL lacking the B1 motif, and caveolin‐1 in B3 LECs was independent of ouabain concentrations, and not seen in all cells. Increase in ouabain concentration enhanced the interaction of NKA with α‐tubulin, which was attenuated by preventing its deacetylation with Trichostatin A, a histone deacetylase inhibitor. Colocalization of NKA with EEA1 was not affected by ouabain, which appeared to increase NKA/Lamp1 immuno‐colocalization. A model is proposed by which NORC is translocated across the plasma membranes within minutes after its generation. In some B3 cells, by interaction with BclXS, caveolin and α‐tubulin, NORC may be guided to endosomes and, if not recycled back to the plasma membrane, subsequently to lysosomes. The future fate of NORC, especially of a putative nuclear receptor capture of lysosome‐processed ouabain to mediate gene transcriptional upregulation of various pro‐apoptotic and pro‐inflammatory proteins is under study and reported elsewhere at this meeting.Support or Funding InformationWright State University Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long‐term Ouabain treatment promotes ATP1A1‐dependent IL‐6 expression in a fetal human lens epithelial cell line (FHL124)

The cardiac glycoside ouabain (OUA) selectively inhibits Na+/K+‐ATPase (NKA) governing the steady state maintenance of the electro‐chemical gradient of Na+ and K+ across the cell membranes. OUA, following binding to the NAK, activates several growth‐related signaling pathways: the EGFR/Src–Ras–ERK pathway and the PI3K1A–PDK–Akt pathway (Xie and Askari, Eur J Biochem 269(10):2434–2439, 2002). OUA also influences pro‐ and anti‐inflammation (Cavalcante‐Silva et al., Frontiers in Physiology, 8:895, 2017) and cell proliferation depending on OUA concentration and incubation time, and cell type (Kulikov et al., Biochim et Biophys Acta 1768: 1691–1702, 2007 and Nguyen et al. JASN 18 (1): 46–57, 2007). Our group proposed and showed the direct interaction between NKA and BcL2 family proteins, which promote or inhibit apoptosis in human fetal lens epithelial cell line (FHL124) (Lauf et al., Cell Physiol Biochem 31:257–276, 2013; Lauf et al., Am J Physiol. Cell Physiol. 308, C51‐60, 2015). Here, we aimed to study the time dependence of OUA action at a concentration of 10−7M on cell viability and transcriptional gene regulation of various pro‐apoptotic and pro‐inflammatory related genes in FHL124 cells. Cells were incubated with either 1–100 μM OUA in 7 μM DMSO or 7 μM DMSO alone (solvent control) relative to an untreated control for 1, 24 and 48 h. The OUA‐treated cells cultured for 24 h revealed a slightly decreased cell viability, however, 48 h incubation dramatically decreased the number of cells by 75–80% while DMSO alone reduced cell viability by 40–50% compared to untreated cells. Like all epithelial cells, FHL124 cells possess α1 (atp1a1) isoform of NAK. The 5′‐untranslated region (5′UTR) of the atp1a1 gene contains two p53 and three NF‐kB (transcription factors nuclear factor‐kappa‐B) response elements. To test for the functional expression of the p53 and NFKB target genes, we selected mdm2, which negatively regulates the p53 gene and IL‐6 (interleukin‐6), which is induced by NF‐KB. Quantitative polymerase chain reaction (qPCR) method, using TaqMan probes, was employed to measure comparative gene expression of the atp1a1, mdm2, and IL‐6 genes. Cells OUA‐treated for 48 h promoted an increase of the IL‐6 expression by ~eight fold and the atp1a1 expression by two fold, but not the mdm2 expression. These findings suggest that OUA, in addition to utilizing existing signaling pathways mentioned above, may act either in complex with cytosolically internalized NKA (see Stricker et al., this FASEB meeting 2018) or derived therefrom through to be determined gene regulation factors proximal to the NF‐kB response elements. Further studies are aimed to confirm NKA‐dependent IL‐6 expression after long‐treatment with OUA.Support or Funding InformationSupported by the P&amp;T Graduate program and WSU Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.

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.

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

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. 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. 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. 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.

Sulfadiazine modified PDMS as a model material with the potential for the mitigation of posterior capsule opacification (PCO)

Cataract surgery, while the most common surgical procedure performed, leads to posterior capsule opacification in approximately 30% of cases. Transforming growth factor beta 2 (TGF-β2) and matrix metalloproteinases (MMPs) have been shown to play important roles in the cellular processes leading to posterior capsule opacification. Delivery of inhibitors to MMPs may have the potential to inhibit the initial cascade of events that lead to PCO. However, delivery of these molecules via tethering has proven difficult. In this work, sulfadiazine was tethered to polydimethylsiloxane (PDMS) via a polyethylene glycol (PEG) spacer as a potential MMPI mimic. Surface characterization using a variety of methods demonstrated successful modification with the antibiotic. The surfaces were examined with lens epithelial cells to determine their effect on these cellular processes, including cell transdifferentiation and production of extracellular matrix components. The presence of TGF-β2 in the cell culture media was found to stimulate the production of ECM components such as collagen, fibronectin, and laminin, as well as alpha smooth muscle actin (α-SMA), and the migration marker Rho by HLE-B3 and FHL124 cells. In all cases, these effects were decreased but not completely eradicated by the presence of sulfadiazine on the PDMS surfaces. While the level of inhibition necessary for inhibition of PCO in vivo is unknown, these results suggest that IOL surface modification with sulfadiazine has the potential to reduce cellular changes associated with PCO. Furthermore, the results demonstrate for the first time that changes consistent with inhibition of fibrosis may be elicited by surfaces modified with sulfadiazine.

The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

Lens degeneration in Fpr1(-/-) mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. Lens epithelial cell FPR1 may be involved in development and maintenance of the lens. This is the first link between non-hematopoietic expression of FPR1 and an ophthalmologic phenotype. Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1(-/-) mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ~2500 specific binding sites on FHL-124 cells (K(D) ~ 0.5 nm) versus ~40,000 sites on neutrophils (K(D) = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca(2+) flux in FHL 124 cells, consistent with classic G(i)-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice.

MMP2 Activity is Critical for TGFβ2-Induced Matrix Contraction—Implications for Fibrosis

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 (TGFβ) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFβ-mediated PCO formation. 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. Active TGFβ2 (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 TGFβ2-induced matrix contraction. Active TGFβ2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 μM), MMP2 siRNA, and MMP2 neutralizing antibody (4 μg/mL). TGFβ2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 μg/mL). MMP2 plays a critical role in TGFβ2-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.

KCC2a Expression in a Human Fetal Lens Epithelial Cell Line

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K⁺Cl^- (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.

Formyl peptide receptor 1 is functionally expressed on human lens epithelial cells and promotes lens homeostasis (149.19)

Abstract Formyl peptide receptor 1 (FPR1) is a chemoattractant receptor specific for phagocytes, without clearly defined function for non-hematopoietic cells. We found that the lens of Fpr1-/- mice degenerates by 6 months of age without infection or inflammation. Thus, we hypothesized functional expression of FPR1 on lens epithelial cells, the only cell type present in lens. Flow cytometry, qPCR and PTX sensitive fMLF-induced calcium flux assays showed functional FPR1 expression in fetal human lens (FHL-124) cells and in the human lens epithelial cell line SRA 01/04. Despite strong anti-FPR1 binding to lens cells by FACS, similar to that observed for human neutrophils or hFPR1-transfected HEK 293 cells, fluorescent FPR1 ligand fNLFNYK-Fl only poorly bound to FHL-124 cells, with &amp;gt; 80% non-specific binding; by contrast, human neutrophils and hFPR1-transfected HEK-293 cells exhibited &amp;gt;70% specific binding. The KD of fNLFNYK-Fl at neutrophil FPR1 and hFPR1-transfected HEK-293 cells was 2-3 nM, but ~ 0.2 nM in FHL-124 cells. fMLF internalized ~80% of neutrophil FPR1, but only ~25% of FHL-124 FPR1. FPR1 protein in FHL-124 cells resisted siRNA-mediated knockdown, despite strongly reduced mRNA levels, and could only be detected by Western blot using alkali-stripped membranes, suggesting high-molecular complexes of FPR1 and peripheral membrane proteins. The data suggest different conformations of FPR1 in neutrophils versus lens epithelial cells and a key function in lens maintenance.

Prevention of posterior capsule opacification by combination of arsenic trioxide and perfect capsule

Despite recent improvements in IOL design, posterior capsule opacification (PCO) remains a significant clinical problem after cataract surgery. The Perfect Capsule device (Milvella, Ltd., Epping, Australia) permits the introduction and subsequent removal of potentially toxic agents into the closed capsular bag. The present purpose was to detect the effectiveness of arsenic trioxide (As2O3) to cultured human lens cells and cells within the human capsular bag. All experiments were carried out with 2 minutes exposure to As2O3. Effect of As2O3 on FHL124 cells growth was tested by MTT. Changes in cell calcium levels induced by high concentration of As2O3 were measured by real-time fluorometric single-cell digital imaging techniques after Fura-2 incorporation. In vitro human capsular bags were also tested after a sham surgery was performed using the Perfect Capsule device to form a closed system. As2O3 (10, 30, 100, 300, 1000 µmol/L) were used with donor match-pairs treated with medium alone.(n = 3 in all cases).On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence cytochemistry. As2O3 of 100 µmol/L and above inhibited the growth of FHL 124 cells in a dose-dependent manner with 2 minutes exposure (t = 5.217, P < 0.01, IC(50) is 130 µmol/L) As2O3 depleted the calcium store and consequently lead to a loss of calcium signaling.Moreover, 1 × 10(4) µmol/L As2O3 had a moderate effect on the calcium influx pathway, inhibition on the amplitude and rapidity is (43.24 ± 2.98)% (t = 3.134, P < 0.01) and (46.27 ± 6.01)% (t = 3.521, P < 0.01), respectively. No cell left in the vitro human capsular bags after being treated with 1 × 10(4) µmol/L As2O3. As2O3 can eliminate the human lens epithelium cells remained in the capsular bag in cataract surgery, which may help to prevent PCO. The application of As2O3 to cells within the capsular bag for a 2 minute window using the Perfect Capsule system predicts this compound will have therapeutic benefit to humans in vivo.

Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 μM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 μM) induced a time-dependent increase in HIF-1α protein levels. Quercetin (30 μM) was also responsible for a rapid and long-lasting translocation of HIF-1α from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT–PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1α was reversed by addition of excess iron (100 μM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells.