Age-related changes of human lens epithelial cells in vivo.

We examined the density and morphology of lens epithelial cells (LECs) in vivo in a group of normal volunteers and cataract patients by using a newly developed noncontact specular microscope. There was a statistically significant decrease in the cell density of LECs in a group of cataract patients over the age of 80 years. The coefficient of variation of the cell area and the number of large black spots that were observed in the enhanced specular images were not related to aging or cataract formation. Our data indicate that the cell density of LECs decreases after reaching the age of 80, but cataract formation does not affect the cell density or the coefficient of variation of the cell area until the age of 80.

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis.

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

Functional analysis of the promoter and chromosomal localization for human LEP503, a novel lens epithelium gene.

LEP503 is a novel gene product isolated from lens epithelial cells by a subtractive cDNA cloning strategy. It is highly conserved in different vertebrate species and developmentally regulated in postnatal rat lens, suggesting that LEP503 may be an important lens epithelium gene involved in the processes of lens epithelial cell differentiation. The expression of LEP503 is highly restricted to lens epithelial cells in vivo. To investigate the molecular mechanisms regulating the promoter of the human LEP503, we cloned and characterized the promoter of the human LEP503 gene. The transcription start site was localized to a nucleotide C 22 base pairs (bp) 5' of the initiation methionine codon. By reporter gene transfection experiments, we found that approximately 2.5-kb of LEP503 5'-flanking sequence directed high level luciferase activity in human lens epithelial cells; further deletion analysis revealed positive regulatory element between bp -401 and +22. Mutation analysis in each of the seven potential binding sites for transcription factors within the region between -401 and +22 showed that the AP-1 element at -131 and the Sp1 element at -48 are the most important sites for the tissue-specific expression of LEP503. Consistent with lens epithelial cell-restricted expression of LEP503 mRNA, we found that the approximately 2.5-kb 5'-flanking sequence directed high-level promoter activity in lens epithelial cells but not in other cell types. Understanding the LEP503 promoter will allow us to investigate lens epithelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to lens epithelial cells. The LEP503 gene is mapped to human chromosome 1q22, the same location to which zonular pulverulent cataract was previously mapped.

Enhanced disruption of the blood-aqueous barrier and the incidence of angiographic cystoid macular edema by topical timolol and its preservative in early postoperative pseudophakia.

OBJECTIVE: To investigate the effects of timolol maleate with preservative and its preserved (PV) and nonpreserved vehicles (NPV) (benzalkonium chloride) on the blood-aqueous barrier and angiographic cystoid macular edema (CME) in early postoperative pseudophakia. PATIENTS AND METHODS: Patients with ocular hypertension, normal tension glaucoma, and primary open-angle glaucoma who underwent surgery for cataracts. The study included a double-masked trial for timolol, PV, and NPV and a single-masked trial on the effect of diclofenac sodium and fluorometholone acetate on all three. The patients were divided into 6 groups, each of which were simultaneously administered the following different combinations of compounds: timolol and diclofenac (group A), timolol and fluorometholone (group B), PV and diclofenac (group C), PV and fluorometholone (group D), NPV and diclofenac (group E), and NPV and fluorometholone (group F). The 6 groups were then compared using a laser flare cell meter to determine the degree of disruption of the blood-aqueous barrier and fluorescein angiography to investigate angiographic CME. The differences in mean daily fluctuations in intraocular pressure were compared on the preoperative baseline day and for 5 weeks postoperatively. Twice daily administration of 0.5% timolol maleate or the vehicles was started 2 days before surgery, and continued until 5 weeks after surgery. Diclofenac or fluorometholone drops were instilled in the eyes 4 times preoperatively, on the day of surgery, and 3 times daily for 5 weeks postoperatively. RESULTS: The flare amount was higher on the third and seventh days in group B than in group D, but was the same after the seventh day. The incidence of angiographic CME was the same between both groups. These 2 factors were significantly lower in group F. These 2 factors were also significantly lower in the 3 groups that received diclofenac instead of fluorometholone, with no difference among these groups. The intraocular pressure decline was significant in groups that received timolol compared with groups that received PV or NPV. CONCLUSIONS: Timolol and its preservative, benzalkonium chloride, cause disruption of the blood-aqueous barrier in early postoperative pseudophakia and increased incidence of angiographic CME. The concurrent administration of nonsteroidal anti-inflammatory drug such as diclofenac prevents these adverse effects without interfering with the drop in intraocular pressure caused by timolol. The addition of benzalkonium chloride to timolol contributes considerably to these adverse effects. CLINICAL RELEVANCE: The present results suggest the cause of similar complications produced by other antiglaucoma eyedrops containing similar preservatives.

[Acute retinal necrosis with varicella zoster dermatitis in the fellow eyelid].

BACKGROUND: We report a case of acute retinal necrosis with contralateral varicella zoster dermatitis. CASE: The patient, a 61-year-old man, developed acute retinal necrosis in the right eye 1 month after varicella zoster dermatitis in the left eyelid. PROGRESS: Intravenous acyclovir, corticosteroids, and laser photocoagulation were effective without any surgical treatment. CONCLUSION: We suggest that ophthalmoscopic examination of both eyes is needed in cases with varicella zoster dermatitis.

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.

Osmotic response element is required for the induction of aldose reductase by tumor necrosis factor-alpha.

Induction of aldose reductase (AR) was observed in human cells treated with tumor necrosis factor-alpha (TNF-alpha). AR protein expression increased severalfold in human liver cells after 1 day of exposure to 100 units/ml TNF-alpha. An increase in AR transcripts was also observed in human liver cells after 3 h of TNF-alpha treatment, reaching a maximum level of 11-fold at 48 h. Among the three inflammatory cytokines: TNF-alpha, interleukin-1, and interferon-gamma, TNF-alpha (100 units/ml) gave the most induction of AR. Differences in the pattern of AR induction were observed in human liver, lens, and retinal pigment epithelial cells with increasing concentrations of TNF-alpha. A similar pattern of AR promoter response was observed between TNF-alpha and osmotically stressed human liver cells. The deletion of the osmotic response element (ORE) abolished the induction by TNF-alpha and osmotic stress. A point mutation that converts ORE to a nuclear factor-kappaB (NF-kappaB) sequence abolished the osmotic response but maintained the TNF-alpha response. Electrophoretic gel mobility shift assays showed two NF-kappaB proteins, p50 and p52, capable of binding ORE sequence, and gel shift Western assay detected NF-kappaB proteins p50 and p65 in the ORE complex. Inhibitors of NF-kappaB signaling, lactacystin, and MG132 abolished the AR promoter response to TNF-alpha.

Oxidative stress induces differential gene expression in a human lens epithelial cell line.

PURPOSE: To identify differentially expressed genes in a human lens epithelial cell line exposed to oxidative stress. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) differential display was used to evaluate differential gene expression in a human lens epithelial cell line (SRA 01-04) when cells were exposed for 3 hours to a single bolus of 200 microM hydrogen peroxide. Differentially expressed genes were identified through DNA sequencing and a nucleotide database search. Differential expression was confirmed by northern blot and RT-PCR analyses. RESULTS: Using 18 primer sets, 28 RT-PCR products were differentially expressed between control and hydrogen peroxide-treated cells. In stressed cells, mitochondrial transcripts nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 and cytochrome b were downregulated 4-fold. Of the cytoplasmic mRNAs, glutamine cyclotransferase decreased 10-fold, whereas cytokine-inducible nuclear protein, alternative splicing factor 2, and beta-hydroxyisobutyryl-coenzyme A hydrolase increased 2-, 4-, and 10-fold, respectively. Analysis of mitochondrial transcripts in a 24-hour time course showed that NADH dehydrogenase subunit 4 mRNA decreased by 2-fold as early as 1 hour after oxidative stress, whereas the rate of decrease was slower for cytochrome b, cytochrome oxidase III, and 16S rRNA. CONCLUSIONS: Oxidative stress induced specific expressed gene changes in hydrogen peroxide-treated lens cells, including genes involved in cellular respiration and mRNA and peptide processing. These early changes may reflect pathways involved in the defense, pathology, or both of the lens epithelium, which is exposed to oxidative stress throughout life.

The effects of extracellular matrix on cell attachment, proliferation and migration in a human lens epithelial cell line.

Lens capsule consists of several kinds of extracellular matrix (ECM) which may play an important role in cell attachment, migration and proliferation of lens epithelial cells as a basement membrane. We have investigated the effects of ECM on cell attachment, proliferation and migration in a human lens epithelial (HLE) cell line. The HLE cell line, SRA 01/04, which was transfected with large T-antigen of SV40 was cultured in the absence of serum. Culture plates were coated with human type IV collagen, laminin or fibronectin. The number of cells were counted at 30-180 min and 3, 5 and 7 days of culture. The rate of BrdU incorporation was measured to study the cell proliferation. The cell migration was measured 1, 3, 5 and 7 days after seeding cells. Integrins, the receptors of ECM, were also detected using antibodies for the cell membrane antigens (CD49b, CD49c, CD49e) by an immunohistochemical method. Although less than 10% of cells attached to the non-coated plate and 50-60% of cells attached to the ECM-coated plates, there was no difference of cell attachment among each ECM used. The cell attachment was almost complete during the first 30 min of culture. Cell proliferation was not enhanced, but cell survival was aided by culture on the ECM components for up to 7 days. The area of cell attachment enlarged on the ECM-coated plates, whereas no migration was observed on the non-coated plate. These data indicate that ECM is the essential factor for cell attachment and increases migration of HLE cells.

Human lens epithelial cell line.

Although primary cultures of human lens epithelial (HLE) cells provide important information concerning the role of epithelium in normal lens and cataract formation, the lack of a cell line precludes a broad range of studies on the metabolism and molecular biology of these cells. We have, therefore developed an HLE cell line. Primary cultures of HLE cells were transfected with plasmid vector DNA containing a large T antigen of SV40. The immortalized cells were characterized with regard to morphology, growth rate, karyotype, and expression of crystallins, aldose reductase and other enzymes. A single clone of the immortalized cells, SRA 01/04, formed a monolayer and grew constantly over 130 passages. Isozyme phenotype showed that SRA 01/04 was of human origin, and the chromosome counts were in the hypotetraploid range. Western blot analysis showed that the cells expressed a very low level of crystallins (alphaA and betaB2) and aldose reductase. Messenger RNA (mRNA) for both alpha and beta crystallins was detected by reverse transcription polymerase chain reaction (RT-PCR) in both early and late passages. Sequence analysis of the PCR products, corresponding to alphaA and betaB2 crystallins in the cell line and in primary cultures of HLE, revealed a 100% match with published human alphaA and betaB2 sequences. These characteristics were unchanged in the cell line in early and late passages. This is the first report of the presence of alphaA and transcripts of mRNA for both alphaA and betaB2 in an established human cell line. This new HLE cell line makes it possible to undertake many future studies on the role of epithelium in lens and cataract formation.

Anti-beta-crystallin antibodies (mouse) or sera from humans with age-related cataract are cytotoxic for lens epithelial cells in culture.

Circulating autoantibodies against lens antigens are prevalent in patients with age-related cataract (ARC), but their pathogenic significance is unknown. We hypothesized that these autoantibodies are cytotoxic for lens epithelial cells (LECs). To test this hypothesis. We incubated LECs with mouse polyclonal or monoclonal antibodies against beta-crystallin (anti-beta) in the presence or absence of guinea pig complement. We found that anti-beta in the presence of the complement bound to and killed mouse LECs (MLECs) and human LECs (HLECs). Sera obtained from patients with ARC also were cytotoxic to both HLECs and MLECs in culture. Heat-inactivated human sera were not cytotoxic to LECs in the absence of the complement, but were cytotoxic to both HLECs and MLECs in the presence of additional complement. These results support the hypothesis that autoantibodies against lens antigens are cytotoxic to LECs, and that cell death may involve complement-mediated pathways.

Peroxide-induced damage in lenses of transgenic mice with deficient and elevated levels of glutathione peroxidase.

Transgenic mice with elevated glutathione peroxidase (GSHPx) activity and gene knockout animals with a deficiency of the enzyme were used to investigate the role of GSHPx in defending the lens against H2O2-induced damage. The effects of peroxide on cultured lenses were determined by using light and transmission electron microscopy to evaluate morphological changes occurring in the epithelium and superficial cortex of the central and equatorial regions of the lens. DNA single-strand breaks in the epithelium were also examined. Following a 30-min exposure to 25 microM H2O2, lenses from normal animals showed distinct changes in the morphology of both the epithelium and superficial cortex. The damage to these cells was extensive in lenses of gene knockout mice in which activity of GSHPx was undetectable. In marked contrast, lenses of transgenic mice, which had 5-fold higher activities of GSHPx, were able to resist the cytotoxic effects. Similar to damage to cell morphology, the extent of DNA strand breaks was significantly lower (40% of control) in H2O2-exposed lenses as compared to normal lenses while DNA damage in gene knockout lenses was 5 times greater than that of GSHPx-rich transgenic lenses. The present studies extend our previous findings on the role of the glutathione redox cycle in the detoxification of peroxide and demonstrate that an increase in GSHPx activity protects the lens against peroxide-induced changes in cell morphology and DNA strand breaks.

A study of growth factor receptors in human lens epithelial cells and their relationship to fiber differentiation.

Recent studies have demonstrated that several growth factors enhance fiber differentiation in cultured human lens epithelial (HLE) cells in early passages. However, these effects gradually decrease in cells of later passages. The purpose of this investigation is to test the hypothesis that the decreasing effect of growth factors on fiber differentiation in later passages may be due to a decrease or the inactivation of growth factor receptors as a function of serial subcultures. Specimens of HLE cells were obtained from infants. First through to fourth passage cells were treated with 10 ng ml-1 of epidermal growth factor, basic fibroblast growth factor or insulin-like growth factor-I. Fiber differentiation was determined from spontaneous lentoid formation by phase-contrast and transmission electron microscopy. Growth factor binding to the receptor on the cell surface was determined by transmission electron microscopy using the conjugates of colloidal gold and growth factors, and the number of receptors on the cell surface were also quantified by immunocytochemistry. Spontaneous lentoid formation was enhanced by all of the growth factors studied in the first passage. However, in the second and third passage only double layering of cells without characteristic fiber differentiation was observed while in the fourth passage, growth factors had no effect on differentiation. The number of growth factor bindings as well as the number of growth factor receptors gradually decreased with the number of passages. The loss of effect of growth factors on fiber differentiation with increasing number of passages correlated with the decrease in receptor number.

Effects of growth factors on proliferation and differentiation in human lens epithelial cells in early subculture.

PURPOSE: A successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance. This study examines the effects of four different growth factors on proliferation and differentiation in HLE cells in early subcultures. METHODS: Specimens of HLE cells were obtained from infants. First- or second-passage cells were cultured in the presence of 10(-2) to 10(2) ng/ml acidic and basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF-I). Cell proliferation was determined from cell number, and fiber differentiation was assessed from the time of appearance, the number of lentoids formed, and the expression of gamma-crystallin. RESULTS: Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater than 10(-1) ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on proliferation appeared only at a concentration of 10(2) ng/ml. EGF, bFGF, or IGF-I at 10 ng/ml affected the time of appearance and the number of lentoids formed within 5 to 7 days. In contrast, lentoids were observed after 42 days without the addition of growth factors. Lentoid formation was accompanied by the expression of gamma-crystallin. CONCLUSIONS: EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation in early subcultures.

Membranous outgrowth suggesting lens epithelial cell proliferation in pseudophakic eyes.

PURPOSE: We sought to determine the incidence and structure of membranous outgrowth, which extends from the anterior capsular opening onto the intraocular lens surface in pseudophakic eyes. METHODS: Thirty-four eyes of 31 patients with age-related cataract were prospectively studied. No patient had any abnormality other than cataract. Each patient underwent continuous circular capsulorhexis, phacoemulsification, and implantation within the capsule of a three-piece posterior chamber lens. A slit lamp and specular microscope were used to observe and photograph the intraocular lens surface and anterior capsular opening every day for the first postoperative week, and at days 14, 21, and 28. We counted the number of eyes with the membranous outgrowth and graded the outgrowth according to its shape and length at each postoperative period. RESULTS: In total, 27 of 34 (79%) eyes had the membranous outgrowth from the anterior capsular opening onto the intraocular lens surface. The membrane was first observed on day 3. Three of 34 eyes had the dendritic or fan-shaped structure, which extended less than 0.5 mm from the capsular edge. The membranes were most frequently found on day 7. Twenty-five of 34 eyes had the outgrowth in various grades. After four weeks, no membranes were observed. CONCLUSIONS: The time course and structure of the membranous outgrowth we observed were comparable to those of the outgrowth of lens epithelial cells under tissue culture conditions. The membranous outgrowth may be the result of a transient but active proliferation of human lens epithelial cells onto the intraocular lens surface.

Corneal damage after glass intraocular lens implantation.

We present three cases of corneal damage in two patients after implantation of an anterior chamber, iris-supported, intraocular lens (IOL) with a glass optic and polyamide haptics. We found bullous keratopathy in all three eyes. Penetrating keratoplasty, IOL removal, and anterior vitrectomy were performed in all cases. We believe the bullous keratopathy was caused by long-term iritis and pseudophakodonesis.