Nobuhiro Ibaraki

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.

Pax6 autoregulation mediated by direct interaction of Pax6 protein with the head surface ectoderm-specific enhancer of the mouse Pax6 gene

The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein.

ESCRS Binkhorst Lecture 2002: Pseudophakic preservative maculopathy

&NA; Many antiglaucoma eyedrops are reported to cause cystoid macular edema (CME) in aphakia and pseudophakia. We review 4 clinical and laboratory studies that compare the incidence of CME in early postoperative pseudophakia in eyes that received preserved latanoprost and timolol, nonpreserved timolol, and the preserved and nonpreserved vehicle for these drugs and looked at the morphological damage to cells and the changes in the indicators of cytokine and prostaglandin (PG) synthesis caused by latanoprost and timolol and the preservative benzalkonium chloride. Based on the findings of these studies, which indicate that the preservative causes increased synthesis of PGs and other substances and intensified postoperative inflammation, the term pseudophakic preservative maculopathy is proposed for CME caused by antiglaucoma eyedrops.

Detection of cytotoxic anti-LEDGF autoantibodies in atopic dermatitis

In the last two decades, atopic dermatitis (AD) has been of increasing clinical significance in Japan. Eight-20% of patients with AD developed progressive cataracts (cataract-AD) and lens epithelial cells (LECs) were severely damaged. Lens epithelium-derived growth factor (LEDGF) is a newly isolated survival factor. In the presence of LEDGF, LECs survive well and in the absence of LEDGF, they become highly susceptible to stress. We investigated (1) whether auto-antibody (auto-Ab) to LEDGF is present in sera of AD patients and (2) whether depletion of LEDGF by the auto-Ab kills LECs. In sera from 26 patients with AD using ELISA, we found significantly higher levels of auto-Ab to LEDGF than that in a normal control group. Affinity purified auto-Ab to LEDGF from these sera killed LECs without complement activation. Levels of histamine in the AD group were significantly higher and levels of prostaglandin E2 were significantly lower than in the normal group. However, statistically there are no differences between sera from AD and cataract-AD in levels of Ab to LEDGF, histamine, prostaglandin E2 (PGE2), immunoglobulin E (IgE) and eosinophiles. We speculate that cataract-AD may be induced, in part, by a combination of high levels of serum histamine and eye rubbing which could break the blood-aqueous barrier to allow the entry of Ab to LEDGF into the privileged compartment, thus, reducing LEDGF levels, resulting in damage to LECs, and cataract formation.

Membranous Outgrowth Suggesting Lens Epithelial Cell Proliferation in Pseudophakic Eyes

PURPOSEnWe 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.nnnMETHODSnThirty-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.nnnRESULTSnIn 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.nnnCONCLUSIONSnThe 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.

Inhibitory Effects of Arg-Gly-Asp (RGD) Peptide on Cell Attachment and Migration in a Human Lens Epithelial Cell Line

Posterior capsule opacification (PCO) after cataract surgery is caused by growth of residual human lens epithelial (HLE) cells on the posterior capsule. We have shown that extracellular matrix (ECM) is an essential factor for HLE cell attachment and migration. The purpose of this study was to examine the inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in an HLE cell line. HLE cell line cells (SRA 01/04) that were obtained by transfection of large T antigen of SV40 were cultured in the absence of serum. The culture dishes were coated with type IV collagen, laminin or fibronectin, and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) RGD peptide (0.1, 0.3, 1.0, 2.0 mg/ml) was added to the medium. The number of attached cells was counted after 90 min of incubation, and the inhibitory effects of GRGDSP RGD peptide on cell attachment were calculated. Cell attachment on the fibronectin-coated dishes was inhibited by GRGDSP RGD peptide at concentrations higher than 0.3 mg/ml; the inhibitory rate was 80% at a concentration of 2.0 mg/ml. The inhibition of cell attachment by GRGDSP RGD peptide on laminin-coated dishes appeared only at a concentration of 2.0 mg/ml, whereas no effects were observed on the type IV collagen-coated dishes. The inhibitory effects of GRGDSP RGD peptide on cell migration were measured in medium containing 2.0 mg/ml of GRGDSP RGD peptide after 1, 3, 5 and 7 days of culture. Cell migration was inhibited by GRGDSP RGD peptide from 1 day of culture on the fibronectin-coated dishes and from 5 days of culture on the laminin-coated dishes, whereas no effects were observed on the type IV collagen-coated dishes. GRGDSP RGD peptide inhibited cell attachment and migration on laminin and fibronectin that have RGD sequences. These data suggested that RGD peptide may have the potential to prevent PCO.

A clinicopathologic case report of inflammatory pseudotumors involving the conjunctiva and lung.

BackgroundInflammatory pseudotumors are characterized histopathologically by aggregates of inflammatory lymphocytes, plasma cells, neutrophils, and fibroblasts. We report a rare case of inflammatory pseudotumor involving both the conjunctiva and lung.CaseA 58-year-old man with a 6-year history of pulmonary inflammatory pseudotumor was referred to our hospital for evaluation of conjunctival swelling in the left eye and bilateral iritis.ObservationsThe subconjunctival tumor enlarged slowly, but regressed spontaneously. After partial resection of the subconjunctival and lung tumors, the presence was confirmed of aggregates of chronic inflammatory cells (lymphocytes, plasma cells, neutrophils, fibroblasts) without noncaseating epithelioid granuloma formation. Gene rearrangement testing ruled out malignancy. The patient was treated with oral corticosteroids for fever and primary biliary hepatic cirrhosis. Iritis signs subsided slightly in response to corticosteroids, but persisted. The temporal subconjunctival pseudotumor resolved without recurrence.ConclusionsThis case was compatible histopathologically with inflammatory pseudotumor, and is a rare case of simultaneous occurrence in the lung and conjunctiva. Jpn J Ophthalmol 2004;48:573–577

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.

Anterior capsule opacification: a cell culture model.

Abstract We cultured the human lens epithelial cells with attaching anterior lens capsule using surgically removed capsular flaps obtained in intraocular lens implantation. The capsular flap was placed with cell side down in a small culture well. In phase contrast microscopy, an anti‐meniscus plate was placed on the bathing medium to avoid optical aberration. The human lens epithelial cells showed outgrowth which ceased after 3 to 4 weeks of incubation. The cells under the capsule lost distinctive cell margin. The cells outgrew from the capsular edge both onto the anterior capsular surface and the well bottom. The outgrowth length and Bromodeoxyuridine uptake indicated a low proliferative potency of the human lens epithelial cells.

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.