Sulforaphane can protect lens cells against oxidative stress: implications for cataract prevention.

PURPOSE: Protecting the lens against oxidative stress is of great importance in delaying the onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide cytoprotection against oxidative stress. We therefore tested the ability of SFN to perform this role in lens cells and establish its ability to delay the onset of cataract. METHODS: The human lens epithelial cell line FHL124 and whole porcine lens culture systems were used. The ApoTox-Glo Triplex Assay was used to assess FHL124 cell survival, cytotoxicity, and apoptosis. The MTS assay was used to assess cell populations. To determine levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess level of gene expression, an Illumina whole-genome HT-12 v4 beadchip was used. Protein expression was determined by Western blot and immunocytochemistry. RESULTS: Exposures of 30 µM H2O2 to FHL124 cells caused a reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24-hour pretreatment with 1 µM SFN. In addition, 1 µM SFN significantly reduced H2O2-induced DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole-genome HT-12 v4 beadchip microarray data revealed eight genes upregulated following 24-hour exposure to 1- and 2-µM SFN, which included NQO1 and TXNRD1. This pattern was confirmed at the protein level. Nrf2 translocated to the nucleus in response to 0.5- to 2.0-µM SFN exposure CONCLUSIONS: The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.

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.

TGFbeta/Smad4-dependent and -independent regulation of human lens epithelial cells.

PURPOSE: Transforming growth factor (TGF)-beta can mediate fibrotic responses via Smad4-dependent and -independent signaling pathways. TGFbeta-induced transdifferentiation of lens epithelial cells to myofibroblasts and matrix contraction contribute to posterior capsule opacification (PCO) after cataract surgery. The primary objective of the study was to determine the importance of Smad4 in TGFbeta2-induced transdifferentiation, matrix contraction, and Smad signaling by human lens epithelial cells. METHODS: The human lens cell line FHL 124 was used. Smad4 knockdown was achieved by using siRNA technology and efficiency tested at the message and protein level by real-time PCR and Western blots, respectively. Smad4 and Smad2/3 nuclear distribution after TGFbeta2 stimulation (10 ng/mL) was determined by immunocytochemistry. Gene expression of transdifferentiation markers, alpha-smooth muscle actin and fibronectin and the inhibitory Smad, Smad7, in the presence and absence of TGFbeta2 (10 ng/mL) was determined with real-time PCR. TGFbeta2-induced contraction was assessed with a patch contraction assay. Suspended bead array technology was used to determine pERK, pP38, and pJNK levels in response to TGFbeta2 over a 2-hour time course. RESULTS: SiRNA targeted against Smad4, successfully reduced expression of Smad4 message and protein after both the initial transfection period and removal of transfection conditions. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 after siSmad4 treatment relative to control siRNA in both the presence and absence of 10 ng/mL TGFbeta2. Smad4 knockdown cells exhibited TGFbeta2-induced Smad2/3 nuclear translocation which did not differ significantly from Smad4-expressing cells. Real-time PCR analysis showed significant induction of alphaSMA and fibronectin transcript in the Smad4-expressing cell line (control siRNA group). These inductions were suppressed in Smad4-knockdown cells. TGFbeta-induced mSmad7 expression in FHL 124 cells was unaffected by Smad4 knockdown. Smad4-expressing cells did not exhibit a significant contractile response after 24 hours of culture in the presence or absence of 10 ng/mL TGFbeta2. However, Smad4-knockdown cells demonstrated a significant reduction in patch area (i.e., contraction), after TGFbeta2 treatment. Bead array analysis showed that TGFbeta2 significantly increased pERK and pP38 levels relative to unstimulated control. No significant change was observed with pJNK. CONCLUSIONS: Smad4 is critical for the expression of transdifferentiation markers, alphaSMA, and fibronectin; in contrast, Smad4 signaling is not essential for TGFbeta-induced Smad7 expression. Smad4 does not appear to be necessary for TGFbeta-induced matrix contraction. TGFbeta does activate Smad-independent pathways in human lens epithelial cells. Therefore, TGFbeta2 regulation of transdifferentiation and matrix contraction appears to be mediated by distinct TGFbeta signaling pathways.

Temporal changes in MMP mRNA expression in the lens epithelium during anterior subcapsular cataract formation.

Transforming growth factor beta (TGFbeta) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFbeta1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFbeta for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (alpha-SMA) immunoreactivity that was evident following 4 days of treatment with TGFbeta. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFbeta treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.

TGF beta-induced contraction is not promoted by fibronectin-fibronectin receptor interaction, or alpha SMA expression.

PURPOSE: Transforming growth factor (TGF)-beta is a potent inducer of both transdifferentiation and contraction, which are regarded as critical processes that underpin tissue fibrosis. Consequently, transdifferentiation is believed to drive TGFbeta-mediated contraction. This study was conducted to determine the relationship between transdifferentiation of human lens epithelial cells and matrix contraction. METHODS: Real-time PCR was used to investigate gene expression of transdifferentiation markers in the human lens cell line FHL 124 and native lens epithelia. Contraction was assessed with a patch-contraction assay, whereby all areas covered by cells were measured with imaging techniques after fixation and cell staining with Coomassie blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To prevent fibronectin-fibronectin receptor interaction 100 microM RGDS peptide was used. Suppression of TGFbeta-induced alphaSMA expression was mediated by siRNA technology. RESULTS: Real-time PCR analysis showed 10 ng/mL TGF-beta1 or -beta2 significantly increased expression of alphaSMA, fibronectin, and alpha5beta1 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGFbeta and RGDS showed a marked increase in the rate of contraction relative to TGF-beta alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and alphaSMA protein when transfected with siRNA. alphaSMA knockdown did not prevent TGFbeta-induced contraction. CONCLUSIONS: A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFbeta-induced matrix contraction.

Arsenic trioxide initiates ER stress responses, perturbs calcium signalling and promotes apoptosis in human lens epithelial cells.

Identification of novel agents to eradicate the residual lens cell population following cataract surgery provides one mode of preventing PCO formation. The present study investigated the biological mechanism of As(2)O(3) cytotoxicity in a human lens cell line and capsular bag system. FHL 124 cell survival was assessed by quantification of total protein content, a cell population measure. Gene changes were detected by Real-time PCR; apoptosis by TUNEL assays. Intracellular calcium was measured by real-time fluorimetric single-cell digital imaging techniques after Fura-2 incorporation. In vitro human capsular bags were generated from donor eyes, which involved sham cataract surgery then use of the Perfect Capsule device to form a closed system to deliver As(2)O(3) for 2min. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence immunocytochemistry. FHL 124 cells demonstrated a dose-dependent sensitivity to As(2)O(3) exposure. A 2min exposure of As(2)O(3) to cells within the capsular bag, using the perfect capsule system, resulted in total cell death when used at 100mM. As(2)O(3) provoked an ER stress response identified through an upregulation of known genes. As(2)O(3) depleted the calcium store and consequently lead to reduced calcium signalling. As(2)O(3) increased rates of apoptosis. Arsenic trioxide provokes ER stress that leads to down-regulation of calcium signalling resulting in apoptosis. The application of As(2)O(3) to cells within the capsular bag for a 2min window using the Perfect Capsule system predicts putative therapeutic benefit in vivo.

Relative suppression of the sodium-dependent Vitamin C transport in mouse versus human lens epithelial cells.

Vitamin C is a major antioxidant and UV absorbent in the human lens. In the rodent lens, the levels are very low for unknown reasons. Searching for clues to explain this suppression, we investigated the comparative uptake of Vitamin C in cultured human and mouse lens epithelial cells. When compared to human HLE-B3 lens epithelial cells, (14)C-ASA uptake was 4- to 10-fold impaired in confluent mouse lens 17EM15 (p < 0.0001) and 21EM15 (p < 0.001) cells, respectively. High glucose concentrations reduced the uptake by 30-50% in all cells (p < 0.005). Incubation of cells with 6-deoxy-6-fluoro-ascorbic (F-ASA), i.e. a probe specific for the sodium-dependent Vitamin C uptake (SVCT2), revealed a 10-fold uptake suppression into mouse 17EM15 relative to human HLE-B3 and JAR choriocarcinoma cells (a control), that could be overcome by overexpressing hSVCT2 using two different promoter constructs. The relative Vitamin C uptake differences suggest either low expression of SVCT2, molecular differences between the transporters themselves or their biological regulation, since a recent study has shown that exogenous feeding of ascorbic acid to rats increased only modestly lenticular uptake (Mody et al., Acta Ophthalmol Scand 83: 228-223, 2005). Elucidation of the mechanism by which SCVT2 activity is suppressed in mouse lens may help unravel a major question of evolutionary significance for night vision in nocturnal animals.

Growth factor receptor signalling in human lens cells: role of the calcium store.

In the human lens, stimulation of tyrosine-kinase coupled growth factor receptors such as epidermal growth factor receptor (EGFR) can induce calcium release from endoplasmic reticulum (ER) stores. The present study investigated the impact of calcium store inactivation on EGFR signalling, cell growth and death in a well-characterised human lens cell line (FHL124). FHL124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% foetal calf serum (FCS) and seeded on 24-well plates (DNA and protein synthesis), tissue culture dishes (growth assay, western immunoblot), and glass coverslips (immunocytochemistry). DNA and protein synthesis rates were quantified by measuring the incorporation of (3)H-thymidine and (35)S-methionine into FHL124 cells in serum-free EMEM or EMEM supplemented with thapsigargin (Tg) (100 nM and 1 microM). Longer-term growth was assessed by quantifying the increase in area over time of a circular patch of seeded cells. EGFR was identified using anti-EGFR mouse monoclonal antibody and visualised by fluorescence microscopy with ALEXA 488 conjugated secondary antibody. Programmed cell death was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay method. Activation of the mitogen-activated protein kinase (MAPK) signalling protein extracellular signal-regulated kinase (ERK) and the cell cycle proteins CDK2 and P27(kip1) were detected by western immunoblot techniques. Inactivation by > or =100 nM Tg inhibited both protein and DNA synthesis although the effect on the latter was greatest. The cell cycle activator CDK2 was reduced by Tg, while the inhibitor P27(kip1) was increased along with the percentage of apoptotic cells. A single, maximal epidermal growth factor (EGF) (10 ng ml(-1)) exposure induced receptor internalization and increased ERK phosphorylation. Both internalisation and ERK activation were unaffected by the presence of Tg. However, reduced internalisation and ERK activation followed repeated EGF applications in the presence of Tg. Additionally, ERK activation by submaximal EGF concentrations was reduced by store depletion. An intact endoplasmic reticulum calcium store therefore plays a significant role in human lens cell survival and growth.

Propyl gallate is a superoxide dismutase mimic and protects cultured lens epithelial cells from H2O2 insult.

n-Propyl gallate (nPG) is a food preservative that is generally regarded as safe by the US FDA. It suppresses oxidation in biological systems. The mechanism by which nPG acts in biological systems is uncertain. We investigated whether nPG protected cultured lens epithelial cells from H(2)O(2)-induced damage. Cells were treated with H(2)O(2) or with nPG and then H(2)O(2). H(2)O(2) inhibited growth, caused membrane blebbing, decreased lactate production, increased the level of GSSG, decreased the levels of GSH, ATP and NAD(+), and G3PDH activity, stimulated the hexose monophosphate shunt and induced single-strand breaks in DNA. nPG prevented the H(2)O(2)-induced growth inhibition, membrane blebbing, drop in NAD(+) and single-strand breaks in DNA. The mechanism by which nPG acts at the chemical level was investigated using electron paramagnetic resonance (EPR), direct spectrophotometric kinetic measurements, and cyclic voltammetry. When nPG at low concentrations (nM to microM) was mixed with a large excess of O(2)(-)*, the superoxide signal was destroyed as indicated by UV visible spectroscopy and EPR. Kinetic analysis indicated that nPG dismutated O(2)(-)* in repetitive additions of superoxide with little loss of activity. The rate constant for the overall reaction of nPG with O(2)(-)* was ca. 10(6)M(-1)s(-1). nPG had a very low specific binding constant for Fe(2+) as determined by cyclic voltammetry. The evidence indicates that nPG dismutates the superoxide ion in a catalytic manner.