Oligonucleotide microarray analysis of human lens epithelial cells: TGFbeta regulated gene expression.

PURPOSE: Transforming growth factor beta (TGFbeta), a pro-fibrotic cytokine has been proposed a causative factor in the progression of lens pathologies including posterior capsule opacification (PCO), a condition that occurs after cataract surgery. This study employs oligonucleotide microarrays to provide a global profile of gene expression in FHL 124 cells, to identify changes in gene expression following treatment with TGFbeta1 and TGFbeta2, and to enable putative genes relating to TGFbeta regulation and PCO to be identified. METHODS: Routinely cultured FHL 124 cells maintained in serum free Eagle's Minimum Essential Medium (EMEM) were treated with either TGFbeta1 or TGFbeta2 at 10 ng/ml for 24 h then total RNA extraction was carried out. Total RNA (16 microg) was used to analyze gene expression by spotted oligonucleotide microarray hybridization. The spotted oligonucleotide microarrays employed contained 13,971 oligonucleotide probes, each designed to be specific for an individual gene. Array images were analyzed using GenePix Pro 3.0, followed by raw data import into GeneSpring 7.0 where a cross gene error model (CGEM) filter was applied. Data was subjected to LoWess normalization prior to comparison of the different treatment groups. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to validate the oligonucleotide microarray data, using a select number of genes exhibiting differential expression. RESULTS: A total of 301 genes were up-regulated by more than 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these up-regulated genes had biological functions relevant to lens epithelial cells including roles in contraction, transdifferentiation and as extracellular matrix (ECM) components. A total of 164 genes were down-regulated by more that 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these down-regulated genes have biological functions including roles in apoptosis, signaling, and as anti-oxidants. Following treatment with TGFbeta1 and TGFbeta2, QRT-PCR successfully validated the differential changes in gene expression detected by oligonucleotide microarrays. CONCLUSIONS: TGFbeta1 and TGFbeta2 regulate the gene expression of genes that have important roles in human lens epithelial cell biology. Most importantly, TGFbeta induces the gene expression of a number of fibrotic markers which may have a role in promoting the development of PCO such as transdifferentiation markers, contractile factors, and ECM components.

TGFbeta2 influences alpha5-beta1 integrin distribution in human lens cells.

TGFbeta plays a central role in posterior capsule opacification, in which cell proliferation and matrix deposition, accompanied by capsular wrinkling, are largely responsible for the increased light scatter involved. Human FHL124 cells were plated onto uncoated glass coverslips to form circular patches so that the central cells reached confluency while the peripheral cells grew outwards. Cell patches were exposed to serum free (SF) EMEM (control) or TGFbeta supplemented (10 ng ml(-1)) EMEM. Fibronectin (Fn), alpha5beta1 integrin and F-actin were localized by immunofluorescence techniques and analysed by confocal microscopy. In the confluent, central cells in SF medium alpha5beta1 showed a punctate distribution while Fn was present in strongly staining fibres. TGFbeta had no effect on integrin or Fn distribution in confluent cells. In the peripheral, motile cells of the patches in SF conditions alpha5beta1 was localized in well-defined focal adhesion plaques at the ends of actin stress fibres, while Fn was distributed in a punctate perinuclear pattern. TGFbeta had a profound dispersing effect on the integrin causing a widespread distribution of alpha5beta1 in the membrane with no apparent association with the actin filaments. The cells had a more fibroblastic morphology with increased deposition of Fn near the nucleus. All the TGFbeta-induced changes were inhibited by the TGFbeta antibody CAT152 (Cambridge Antibody Technology). Culture with a function-blocking alpha5 antibody or Fn antibody resulted in detachment of the peripheral cells from the patches, but the central cells remained intact. The patch culture method therefore provides a convenient means of investigating the differences between confluent and growing lens cells both in terms of the patterns of alpha5beta1 integrin and Fn and also in the response of the molecular arrangements of both to TGFbeta2.

Characterisation of TGF-beta2 signalling and function in a human lens cell line.

There is increasing evidence implicating Transforming growth factor beta (TGF-beta) in pathological states of the lens. However, the underlying signalling mechanisms in human cells have not been fully examined. We have therefore investigated in a human lens cell line, FHL 124, the signalling characteristics of TGF-beta and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-beta2 antibody, CAT-152, in suppressing TGF-beta2 induced changes in a number of conditions. FHL 124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FCS. Characterisation of the cell line was determined using Affymetrix gene microarrays and compared to native human lens epithelium. Cells were serum starved for 24 hr prior to exposure to TGF-beta2 in the presence and absence of CAT-152. Non-stimulated cells served as controls. Smad 4 localisation was observed by immunocytochemistry. To study Smad-dependent transcriptional activity, cells were transfected with SBE4-luc, an artificial smad-specific reporter, using Fugene-6. Transcriptional activity was determined by luciferase activity. Gene expression was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Proliferation was determined by 3H-thymidine DNA incorporation. Growth and contraction were assessed using a scratch and patch assay. Affymettrix gene microarrays identified 99.5% homology between FHL 124 cells and the native lens epithelium with respect to expression pattern of the 22,270 genes on the chip. Moreover, FHL 124 cells expressed phenotypic markers, alphaA-crystallin and pax6 along with lens epithelial cell specific marker FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-beta2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-beta2 enhanced the expression of mRNA levels of alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Exposure to TGF-beta2 resulted in a relatively small inhibition of 3H-thymidine incorporation of FHL 124 cells. However, a more marked contractile effect was also observed. In serum-supplemented medium, growth rates and TGF-beta induced contraction were enhanced. Treatment with 0.1-10 microg ml(-1) CAT-152 dose-dependently inhibited 10 ng ml(-1) TGF-beta2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-beta therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-beta2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-beta induced contraction is enhanced. The work presented therefore demonstrates a platform technology to study TGF-beta2 signalling in human lens epithelial cells and provides evidence to show TGF-beta2 can be a potent factor in the development of posterior capsule opacification following cataract surgery.

Stress-induced ATP release from and growth modulation of human lens and retinal pigment epithelial cells.

ATP release has been shown to occur following stimulation in several cellular systems. This study was undertaken to determine if lens and retinal epithelial cells release ATP in response to physiological stresses and to elucidate a possible role for ATP. Analysis of human aqueous humour samples showed a mean ATP level of 37.8+/-7.7 nM. Hyper-osmotic stress induced a dose- and time-dependent release of ATP. Both cell types were found to proliferate in serum-free medium, and the addition of ATP and adenosine at concentrations as low as 0.1 nM inhibited growth. Gene profiling also demonstrated the presence of the ectonucleotidases CD39 and CD73 and the A1 adenosine receptor on both cell types.

Ubiquitin-activating enzyme (E1) isoforms in lens epithelial cells: origin of translation, E2 specificity and cellular localization determined with novel site-specific antibodies.

Lens development and response to peroxide stress are associated with dramatic changes in protein ubiquitination, reflecting dynamic changes in activity of the ubiquitin-activating enzyme (E1). Two isoforms of E1 (E1A and E1B) have been identified in lens cells although only one E1 mRNA, containing three potential translational start sites, has been detected. Novel, site-specific antibodies to E1 were generated and the hypothesis that the two isoforms of E1 are translated from alternative initiation codons of a single mRNA was tested. Antibodies raised against E1A-N peptide (Met(1)to Cys(23)of E1A) reacted only with E1A by immunoblot and immunoprecipitation. Antibodies raised against E1B-N peptide (Met(1)to Glu(25)of E1B or Met(41)to Glu(65)of E1A) and E1AB-C peptide (His(1030)to Arg(1058)of E1A or His(990)to Arg(1018)of E1B) reacted with both E1A and E1B. These results indicate that (1) E1A and E1B contain the same C-terminal residues; (2) E1A contains the N terminal sequence of E1B; and (3) E1B does not contain the N terminal sequence of E1A. The two isoforms of lens E1 are therefore translated from a single mRNA. Specifically, E1A is translated from the first initiation codon, and E1B translated from the second initiation codon. E1A and E1B were affinity-purified, and their ability to 'charge' ubiquitin carrier proteins (E2s) with activated ubiquitin was compared in a cell-free system. E1A and E1B were indistinguishable with respect to charging different E2s. However, E1 immunolocalization studies with human lens epithelial cells indicate that E1A and E1B are preferentially localized to the nucleus and cytosol, respectively. This observation suggests that E1A and E1B ubiquitinate different proteins and serve different functions in intact cells.

Hepatocyte growth factor function and c-Met expression in human lens epithelial cells.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor c-met perform a multitude of functions. However, despite the significant degree of study of HGF and c-met in numerous tissues and cell types, relatively few investigations have been performed on the lens. In the current study, therefore, the role of HGF and the receptor c-met in human lens epithelial cells was investigated. METHODS: Anterior epithelium and capsular bags were prepared from human donor eyes and maintained in Eagle's minimum essential medium (EMEM) in a 5% CO(2) atmosphere at 35 degrees C. In addition, the human lens cell line FHL124, was routinely cultured and seeded onto glass coverslips (c-met immunodetection), 12-well plates (DNA and protein synthesis), and tissue culture dishes (migration). c-Met was detected by immunocytochemistry and fluorescence-activated cell scanning (FACS). HGF was measured using enzyme-linked immunosorbent assay (ELISA) techniques. Proliferation and protein synthesis were determined by [(3)H]thymidine and (35)S-methionine incorporation into DNA and proteins, respectively. Migration was assessed using a scratch-wound assay and time-lapse video microscopy. RESULTS: HGF was detected at all stages of culture of capsular bags in protein-free medium. Moreover, c-met was present on the native epithelium and after mechanical trauma was seen to be upregulated. Immunolocalization and FACS analysis demonstrated c-met expression on FHL124 cells throughout the whole population. Furthermore, FACS analysis showed that serum-maintained cells sustained a higher level of receptor expression relative to serum-deprived cells. Additionally, HGF was found to stimulate proliferation, protein synthesis, and migratory responses. CONCLUSIONS: c-Met receptors are expressed in native epithelium, capsular bag cultures, and FHL124 cells. Receptor is distributed across the entire cell population; however, this expression is environmentally and mechanically sensitive. HGF is also present in capsular bags at all stages of culture. In addition, HGF can stimulate migration, proliferation, and protein synthesis. It therefore appears that a multifunctional autocrine loop involving HGF and c-met is in place and could be important in the development of posterior capsule opacification.

Induction of heme oxygenase-1 modulates cis-aconitase activity in lens epithelial cells.

Heme oxygenase-1 is the heme catabolic enzyme induced in human dermal fibroblasts by environmental stress. We report an increase of heme oxygenase-1 message in lens epithelial cells after exposure to UVA radiation, followed by a 10-fold increase of protein expression. The size of message was larger than previously demonstrated for fibroblasts. The relationship between heme oxygenase-1 activation and iron metabolism was investigated by measurement of activities of both cytosolic and mitochondrial cis-aconitase enzymes. A 2-fold increase in mitochondrial cis-aconitase activity in UVA-exposed cells coincided with the time of maximal heme oxygenase-1 expression. We propose that modulation of cis-aconitase activity at the translational level by an increase of cellular iron is an important consequence of heme oxygenase-1 activation. This might be a novel aspect of the protective role of heme oxygenase-1 in modulating the response of cells challenged with oxidative stress.

Protection from oxidative insult in glutathione depleted lens epithelial cells.

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2 and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2O2-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H2O2 when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15-20% of the control values without a concomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to an initial concentration of 0. 05 mm H2O2 (for 50,000 cells) or 2 doses of 0.5 mm H2O2 (for 800,000 cells). After exposure to H2O2, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3 and 6 and counted on day 7. When 50,000 or 800,000 cells with decreased glutathione were exposed to 0.05 or 0.5 mm H2O2 the H2O2 was cytotoxic, whereas cells treated with H2O2 alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H2O2 from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H2O2 exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EM level. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells were treated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine and then challenged with H2O2 cytotoxicity was prevented and the cells were capable of growth. The data show that H2O2 was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the generation of reactive oxygen species. In addition, the results indicate that GSH has an important protective role independent of its ability to decompose H2O2 via glutathione peroxidase.

Thioltransferase is present in the lens epithelial cells as a highly oxidative stress-resistant enzyme.

The redox homeostasis is controlled by several enzyme systems. Sulfhydryl groups in lens proteins are very sensitive to oxidative stress and can easily conjugate with nonprotein thiols (S-thiolation) to form protein-thiol mixed disulfides. We have observed an elevation of protein S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) in cataractous lenses from humans and from animal models subjected to oxidative stress. We also observed that these protein-thiol mixed disulfides could be spontaneously dissociated and lowered to basal levels if the lens which was pre-exposed to H2O2 was subsequently cultured in H2O2-free medium. This suggests that the lens has a system to repair oxidative damage through dethiolation thereby restoring its redox homeostasis. In other tissues, an enzyme, thioltransferase (TTase), has been shown to be responsible for thiol/disulfide regulation. We recently demonstrated the presence of this enzyme in the lens and in cultured lens epithelial cells. Here, we investigated the response of TTase to H2O2 stress and its possible repair function in cultured lens epithelial cells. Rabbit lens epithelial cell line N/N 1003A was raised to confluence, trypsinized and plated at 0.8 million cells per 60 mm culture dish. The cells were incubated overnight in Eagle's minimum essential medium (MEM) with 1% rabbit serum and then in serum-free MEM for 30 min before a bolus of 0.5 mm H2O2 was added. At intervals of 5, 15, 30 min and up to 3 hr, the cells were harvested and used for enzyme assays for TTase, glutathione reductase (GR), glutathione peroxidase (GPx) and glyceraldehyde-3-phosphate dehydrogenase (G-3PD). Free GSH, total SH and PSSG and PSSC were also determined. Hydrogen peroxide in the medium was measured at each time point. Cells incubated without H2O2 were used as controls. The results showed that the H2O2 concentration was reduced to 50% within 30 min and was undetectable at 2 hr. Cellular GSH dropped to 40% within 5 min and stayed at this level before it began to increase at 90 min and completely recovered by 2 hr. The total SH groups were similar to free GSH. PSSG and PSSC increased 6.5 and 2 times respectively before 30 min and then decreased when GSH started to recover. G-3PD was most sensitive to H2O2 and lost 95% activity within 5 min. The activity was regained quickly when H2O2 diminished in the medium. A similar but less severe pattern was observed in both GPx (60% loss at 60 min) and GR (30% loss at 90 min). In contrast, TTase activity remained constant during the entire 3 hr. Only when a higher dose of H2O2 (0.8-1.0 mM) was used, did TTase activity show a brief loss (<30% at 60 min) and a swift recovery. Cells exposed to H2O2 exhibited a normal morphology with no evidence of DNA fragmentation. The lens epithelial cells showed a remarkable ability to repair the early damages induced by H2O2. The unusual oxidative stress-resistant property displayed by TTase, coupled with its known function suggest that it plays an important role in the repair of oxidative damage.

Heme oxygenase synthesis is induced in cultured lens epithelium by hyperbaric oxygen or puromycin.

We showed previously that treatment of cultured rabbit lens epithelial cells (LECs) with hyperbaric oxygen (HBO) produced DNA strand-breaks, caused reversible inhibition of protein synthesis and induced the synthesis of a 32 kD protein. In the present work, we employed immunostaining procedures to identify the 32 kD protein as heme oxygenase-1 (HO-1). Increased synthesis of the enzyme was observed as early as 12 hr after HBO-treatment, reached a maximum at 18 hr and was not detectable at 36 hr. Exposure of the cells to hemin also increased the synthesis of HO-1. An HBO-induced inhibition of protein synthesis and the subsequent induction of HO-1 was also observed in the capsule-epithelium of cultured rabbit lenses. For both LECs and the cultured lens, only HO-1 and not heme oxygenase-2 was HBO-inducible. Use of the antioxidant dimethylthiourea with HBO-treated lenses or LECs did not alter the observed effects on protein synthesis or the induction of HO-1. In contrast to results obtained with 50 atm O2, a pressure of 25 atm O2 inhibited protein synthesis only slightly and failed to induce synthesis of the 32 kD protein (although, as shown previously, identical exposure of LECs to 25 atm O2 significantly damaged DNA). Inhibition of protein synthesis in LECs and cultured lenses with the use of puromycin also induced synthesis of HO-1. Both hemin (10 micron), a source of iron, and 50 atm O2 produced a three-fold increase in the concentration of ferritin, a natural iron chelator, in LECs two days after exposure; no effects on ferritin levels were observed after 1 or 3 days. The finding that the increase in ferritin concentration occurred in the cells significantly after hemin- or HBO-induced synthesis of heme oxygenase indicates that chelatable iron rather than the heme molecule itself may have been the primary agent responsible for inducing ferritin synthesis. The data suggest that HBO-induced synthesis of HO-1 in the lens epithelium may be the result of an inhibition of protein synthesis, possibly leading to an accumulation of heme, rather than a direct protective response against oxidative stress.

Expression of Crystallins, Pax6, Filensin, CP49, MIP, and MP20 in lens-derived cell lines.

PURPOSE: Cell lines are the systems of choice to analyze cellular functions related to the particular organ system. For lens research, three cell lines are widely used: N/N1003A (derived from rabbit lenses), alpha TN4, and NKR-11 (both of murine origin). The aim of the current study was to characterize these particular cell lines with respect to their expression of genes that are considered to be lens specific or expressed preferentially in the lens, such as crystallins, Pax6, Filensin, CP49, MIP, and MP20. METHODS: alpha A- and alpha B-crystallin cDNA from rabbit lenses were sequenced. The expression of various genes was analyzed by reverse transcription-polymerase chain reaction using specific primers and mRNA from three lens-derived cell lines. For control, the expression of the selected genes was compared in nonlenticular tissues of mouse as well as in non-lens-derived murine cell lines (EF43, NIH-3T3, and L929). RESULTS: None of the transcripts for beta B2-crystallin, gamma-crystallins, MIP, MP20, filensin, and CP49 could be detected in the lens-derived cell lines. Transcripts for alpha A-crystallin were amplified in alpha TN4, but not in N/N1003A and NKR-11 cells. Pax6, a master control gene of eye development, is expressed in all three lens-derived cell lines and, additionally, in cell lines of neuronal origin, but not in corneal endothelial cells and in the currently used control cell lines. CONCLUSIONS: Three cell lines of lenticular origin were tested for expression of genes that were found abundantly in the lens. The observed expression of Pax6 in all lens-derived cell lines allows their use in the analysis of corresponding signal chains.

Regional differences in the distribution of catalase in the epithelium of the ocular lens.

Oxidative stress is thought to play a major role in cataract formation. The present experiments are aimed at gaining a better understanding of the systems that protect the lens from damage by reactive oxygen species. The aqueous humor normally contains hydrogen peroxide (H2O2), a compound capable of generating reactive oxygen species. The systems protecting the ocular lens from oxidative damage are primarily confined to the epithelium, a single layer of cells on the anterior side of the organ directly beneath the lens capsule. When cultured rabbit lenses were challenged with a single dose of 0.2 mM H2O2, cells in the peripheral region of the epithelium survived; those in the central region died. Here we investigate the histochemical and immunoperoxidase distributions of catalase, an enzyme which detoxifies H2O2, in cells from the peripheral and central regions of the epithelium on flat mount preparations of the epithelium. In a flat mount, the entire population of lens epithelial cells can be viewed on one preparation. The reaction product for catalase activity and its immunoperoxidase localization were more intense in peripheral epithelial cells than in cells throughout the central epithelium. Treatment of cultured lens epithelial cells or rabbit lenses with 3-aminotriazole or potassium cyanide, inhibitors of catalase, reduced or abolished the histochemical reaction product. Ultrastructural cytochemistry confirmed the presence of catalase in microperoxisomes of the epithelial cells from whole lenses. The decreased level of catalase throughout the central epithelium may account for the increased susceptibility of these cells to H2O2-induced cell death.

Enhanced prostaglandin synthesis after ultraviolet-B exposure modulates DNA synthesis of lens epithelial cells and lowers intraocular pressure in vivo.

PURPOSE: To study the functional significance of prostaglandin synthesis after ultraviolet-B (UVB) exposure of cultured human lens epithelial cells and rabbit eyes in vivo. METHODS: Prostaglandin E2 (PGE2) was assayed using a radioimmunoassay (RIA) and mass spectroscopy. An immortalized human lens epithelial cell line (HLE-B3) was exposed to UV irradiation, and the synthesis of PGE2 was compared with the rabbit lens epithelial cell line N/N1003A. Intact human lenses were exposed to UVB in organ culture. [3H]Thymidine incorporation was measured in cultured lens epithelial cells by incubation with the radiolabel. The effects of isobutyl methyl xanthine (IBMX), an inhibitor of phosphodiesterase and of dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, on PGE2 synthesis and DNA synthesis, were determined. Rabbit eyes were exposed to UVB radiation in vivo. Intraocular pressure was measured at specific times after exposure. Aqueous humor was remove from rabbit eyes, and its PGE2 content was measured by RIA. RESULTS: Cultured human lens epithelial cells (HLE), like rabbit lens epithelial cells (RLE), showed a dose-dependent increase in basal PGE2 synthesis 24 hours after UVB exposure. However, the amount of PGE2 synthesis was 2000-fold higher in the rabbit cells. Ultraviolet-B radiation enhanced the incorporation of [3H]thymidine in lens epithelial cells. Pretreatment of cells with indomethacin reduce PGE2 synthesis and [3H]thymidine incorporation. The human and rabbit cells responded in a similar manner to changes in DNA synthesis after UVB exposure. The addition of IBMX or dbcAMP to indomethacin-treated, UVB-exposed cells restored DNA synthesis toward the levels observed in the UVB-exposed cells. An increase in the concentration of cAMP was observed in lens epithelial cells exposed to exogenous PGE2. PGE2 synthesis in intact human lenses also increased twofold 24 hours after UVB exposure. Exposure of the rabbit eye in vivo to an optimal dose of UVB produced an increase in the PGE2 levels of the lens and the aqueous humor. Measurements of the intraocular pressure (IOP) of the animals showed a decrease in IOP by 2.21 +/- 0.66 and 6.45 +/- 0.79 mm Hg (mean +/- SEM, P = 0.004, t-test) at 6 and 24 hours after UVB exposure, respectively. The decrease in IOP was prevented by pretreatment with indomethacin. Exposure of the rabbit lens to UVB radiation in vivo enhanced [3H]thymidine incorporation twofold into the lens. Pretreatment of rabbits with indomethacin before exposure reduced this response. CONCLUSIONS: Results indicate that UVB exposure enhances PGE2 synthesis in HLE cultures as well as in rabbit lenses irradiated in vivo. This increased PGE2 synthesis is related to the increase in DNA synthesis observed after UVB treatment. The modulation of DNA synthesis in cultured lens epithelial cells after UVB exposure may be mediated by a cAMP-dependent mechanism.

Effect of oxidative insult on young and adult cardiac muscle cells in vitro.

The effect of hydrogen peroxide on cultured neonatal and adult cardiac myocytes was investigated. On neonatal cardiac myocytes the effect was very pronounced at a low concentration (0.03 mM), whereas the adult cardiac myocytes were resistant to the same concentration of H2O2. Dividing neonatal cardiac myocytes were more susceptible to H2O2 insult than the non-dividing adult cardiac myocytes. At a concentration of 0.1 mM H2O2, the neonatal cardiac myocytes were significantly damaged compared with the adult cardiac myocytes. Cardiac muscle cells from neonatal and adult hearts at high density culture were more tolerant to the oxidative insult by H2O2 than cells in low density culture. The damaging effect of H2O2 was very selective on F-actin in neonatal and adult cardiac muscle cells. The effect of H2O2 on myosin, titin, alpha-actinin, desmin or tubulin was not pronounced. Microscopical studies suggested a more marked protection by catalase than by glutathione reductase in the neonatal cells.

Calcium mobilisation modulates growth of lens cells.

The modulating effect of calcium cell signalling agonists on tissue growth was studied in a rabbit lens cell line (NN1003A). Calcium mobilisation was measured after Fura-2 incorporation and growth assayed either by direct Coulter counting or [3H]-thymidine incorporation. Transient increases in cytoplasmic calcium were elicited by rabbit serum, histamine, ATP and PDGF. Thapsigargin induced a prolonged increase and all of the above agonists failed to elicit a response after thapsigargin. Rabbit serum and PDGF both increased cell growth in a concentration-dependent manner. While histamine and ATP had little effect in serum-free medium, they reduced serum-stimulated growth. Acetylcholine and FGF did not produce a marked rise in cytoplasmic calcium and neither did they modulate growth. Both thapsigargin and caffeine greatly inhibited growth. These findings indicate that, in lens cells, agonists that mobilise calcium, whether by acting through G-protein or tyrosine kinase receptors, also modulate lens cell growth. Agents such as thapsigargin and caffeine that inactivate the same calcium store also inhibit growth.

Hydrogen peroxide affects specific epithelial subpopulations in cultured rabbit lenses.

PURPOSE: To investigate the effect of hydrogen peroxide on the epithelial cells of cultured rabbit lenses. METHODS: Lenses were cultured in minimum essential medium containing a single dose of 0.03, 0.1, or 0.2 mM H2O2. Three hours later the medium was replaced with peroxide-free minimum essential medium. Lenses were also treated with 0.5 mM 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU) to lower the activity of glutathione reductase and then exposed to 0.03 mM H2O2 maintained nearly constant by glucose oxidase. After H2O2 treatment, lenses were fixed and whole mounts of the epithelium were prepared or lenses were processed for electron microscopy. RESULTS: Cells exposed to a single dose of 0.03 mM H2O2 appeared normal; 0.1 mM H2O2 was not cytotoxic. Exposure to 0.2 mM H2O2 elicited swelling in cells in the pre-equatorial region (30 minutes) followed by the formation of islands of cells in the pre-equatorial region at 1 hour. Central epithelial cells appeared normal at 1 hour, were swollen at 3 hours and dead at 24 hours. By 48 hours, dead cells were found in the pre-equatorial and central regions. Cells in the peripheral region of the epithelium did not exhibit cytotoxicity. If lenses were pretreated with BCNU and then challenged with a maintained level of 0.03 mM H2O2, cytotoxicity was induced in the central and pre-equatorial regions. Cells in the peripheral region survived BCNU-H2O2 treatment. CONCLUSIONS: Cells in the peripheral region of cultured lenses were more resistant to H2O2 cytotoxicity than cells in the central and pre-equatorial regions. The antioxidant defense or repair systems for H2O2-induced damage do not appear to be uniformly distributed in subpopulations of the lens epithelium.

Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.

PURPOSE: To determine if lens epithelial lines can be established from cryopreserved whole rabbit lenses and from cryopreserved capsule-epithelial preparations (CEPs). METHODS: Lenses or freshly isolated CEPs were cryopreserved and subsequently thawed. Thawed whole lenses were cultured for 48 hours in growth medium and fixed, and whole mounts were examined for mitosis. In addition, CEPs were peeled from cryopreserved lenses and placed in tissue culture. Viability of cryopreserved cells was assessed measuring attachment efficiency and growth. RESULTS: Whole mounts from cryopreserved lenses that were thawed and placed in organ culture in a serum-containing medium exhibited numerous mitotic figures. Freshly isolated CEPs that were cryopreserved and CEPs from cryopreserved lenses generated cell lines. Attachment efficiency was 90% within 3 hours of plating. When 50,000 cells from cryopreserved CEPs were cultured in growth medium, 10(6) cells were noted after 7 days of culture. The cells completed 27 population doublings and showed no sign of senescence. CONCLUSIONS: Rabbit lens epithelial cell lines can be initiated from cryopreserved lenses or CEPs.

Lens epithelium: a primary target of UVB irradiation.

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.

Differentiation and angiogenic growth factor message in two mammalian lens epithelial cell lines.

Lens epithelial cells in culture can sometimes be induced to form spheroid aggregates termed lentoid bodies, composed of cells exhibiting various characteristics of the more highly differentiated lens fiber cells. However, lentoid bodies are often slow to form, and the ability to produce them declines with serial subculture. It was therefore of interest to establish and/or characterize lens epithelial cell lines capable of forming lentoid bodies. The differentiation state was assessed in lentoid bodies formed by each of two lens epithelial cell lines, the transformed alpha TN4 cell line from mouse and the nontransformed N/N1135A cell line from rabbit. Lentoid and monolayer cultures of each cell line were examined for transcripts of the lens-specific alpha A-crystallin ("alpha A"), gamma D-crystallin ("gamma D"; formerly gamma 1-crystallin) and MP26 genes. alpha TN4 lentoid bodies contained 2.5 times the alpha A RNA found in monolayer cells, but lacked detectable gamma D and MP26 RNA. None of the three markers were detected in either lentoid or monolayer N/N1135A cultures grown under the conditions described. Lentoid body formation alone, therefore, does not indicate the extent of differentiation occurring. At least some of the changes in cell adhesion occurring during lentoid body formation involve laminin-like and fibronectin-like interactions, and are reminiscent of those observed during embryonic lens formation. Finally, vascular endothelial growth factor mRNA was absent from the lens but present in alpha TN4 cells, suggesting a mechanism whereby the lens tumors of the founder mouse became vascularized.