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Sample records for lens epithelial cell

  1. Control of lens epithelial cell survival

    PubMed Central

    1993-01-01

    We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture. PMID:8491781

  2. Transcytotic passage of albumin through lens epithelial cells.

    PubMed

    Sabah, Judith R; Schultz, Bruce D; Brown, Zach W; Nguyen, Annelise T; Reddan, John; Takemoto, Larry J

    2007-03-01

    To characterize the transcytotic passage of albumin through lens epithelial cells. N/N 1003A rabbit lens epithelial cells were grown to a confluent monolayer on porous filter supports (Transwell Corning, Inc., Corning, NY). Monolayers were exposed apically to Alexa 488-labeled albumin (Alexa 488-BSA) in the absence and presence of endocytic inhibitors (filipin; dansylcadaverine [DCV]). Transcytotic passage of albumin was monitored for 4 hours by quantitating fluorescence in the basolateral compartment. The mechanism of albumin passage was studied by labeling cell monolayers and cryosections of whole rat lenses for clathrin or caveolin. The monolayer of cells formed a barrier to the passage of albumin, as shown by the 44% reduction in albumin passage in comparison to nonseeded membranes. Treatment with filipin or DCV reduced the passage of Alexa 488-BSA through lens epithelial cells by 73% and 66%, respectively. Confocal microscopy showed that albumin passage was predominantly transcellular and demonstrated colocalization of albumin with caveolin-1 and clathrin in lens epithelial and fiber cells. The Transwell apparatus is an excellent system to monitor transport systems across cell monolayers. In this study, rabbit lens epithelial cells formed a confluent monolayer that acted as a barrier to the passive diffusion of albumin. The kinetics of albumin movement across the monolayer and the inhibitor pharmacology suggests that lens cells actively transport albumin from the apical to the basolateral compartment. The inhibitory profile suggests the involvement of caveolae and clathrin-coated vesicles in the transcytotic process.

  3. Cell division and cadherin-mediated adhesion regulate lens epithelial cell movement in zebrafish.

    PubMed

    Mochizuki, Toshiaki; Luo, Yi-Jyun; Tsai, Hsieh-Fu; Hagiwara, Akane; Masai, Ichiro

    2017-02-15

    In vertebrates, lens epithelial cells cover the anterior half of the lens fiber core. During development, lens epithelial cells proliferate, move posteriorly and differentiate into lens fiber cells after passing through the equator. To elucidate the mechanisms underlying lens epithelial cell movement, we conducted time-lapse imaging of zebrafish lens epithelium. Lens epithelial cells do not intermingle but maintain their relative positions during development. Cell division induces epithelial rearrangement, which subsequently promotes cell movement towards the equator. These data suggest that cell division is the major driving force for cell movement. In zebrafish, E-cadherin is expressed in lens epithelium, whereas N-cadherin is required for lens fiber growth. E-cadherin reduced lens epithelial cell movement, whereas N-cadherin enhanced it. Laser ablation experiments revealed that lens epithelium is governed by pulling tension, which is modulated by these cadherins. Thus, cell division and cadherin-mediated adhesion regulate lens epithelial cell movement via modulation of epithelial tension. © 2017. Published by The Company of Biologists Ltd.

  4. Visualizing lens epithelial cell proliferation in whole lenses

    PubMed Central

    Shui, Ying-Bo; Beebe, David C.

    2010-01-01

    Purpose To develop a means to image cells in S-phase of the cell cycle while preserving the anatomic relationships within the lens. Methods Mice were injected with the thymidine analog, EdU. Whole lenses were removed, fixed and permeabilized. Cells that had incorporated EdU into their DNA were chemically labeled using fluorescent azides and “click” chemistry. Double labeling was performed with antibodies to other antigens, like phospho-histoneH3, a marker of mitotic cells. The position of labeled cells and lens anatomy was viewed using a simple device to position and flatten the lens. Results The nuclei of cells in S-phase of the cell cycle were intensely stained without the use of antibodies. Stained cells were readily localized with reference anatomic landmarks, like the transition zone. Whole lenses could be assayed by rotating the lens on the microscope stage. Double-labeling permitted the co-localization of markers in cycling cells. Conclusions EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens. PMID:20664699

  5. Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in lens epithelial cells.

    PubMed

    Pintwala, Robert; Postnikoff, Cameron; Molladavoodi, Sara; Gorbet, Maud

    2015-03-01

    Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and production of reactive oxygen species via the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate were examined in macrophages. α-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation (p < 0.03) versus control (no intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive oxygen species production in macrophages after one day (p < 0.03) and increased expression of α-smooth muscle actin in HLE B-3 after six days, although only poly(2-hydroxyethyl methacrylate) induced a significant difference versus control (p < 0.01). Our results imply that-contrary to prior uveal biocompatibility understanding-macrophage adherence is not necessary for a strong inflammatory response to an intraocular lens, with hydrophilic surfaces inducing higher activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal

  6. Nanoceria have no genotoxic effect on human lens epithelial cells

    NASA Astrophysics Data System (ADS)

    Pierscionek, Barbara K.; Li, Yuebin; Yasseen, Akeel A.; Colhoun, Liza M.; Schachar, Ronald A.; Chen, Wei

    2010-01-01

    There are no treatments for reversing or halting cataract, a disease of the structural proteins in the eye lens, that has associations with other age-related degenerative conditions such as Alzheimer's disease. The incidence of cataract and associated conditions is increasing as the average age of the population rises. Protein folding diseases are difficult to assess in vivo as proteins and their age-related changes are assessed after extraction. Nanotechnology can be used to investigate protein changes in the intact lens as well as for a potential means of drug delivery. Nanoparticles, such as cerium oxide (CeO2) which have antioxidant properties, may even be used as a means of treating cataract directly. Prior to use in treatments, nanoparticle genotoxicity must be tested to assess the extent of any DNA or chromosomal damage. Sister chromatid exchanges were measured and DNA damage investigated using the alkaline COMET assay on cultured human lens epithelial cells, exposed to 5 and 10 µg ml-1 of CeO2 nanoparticles (nanoceria). Nanoceria at these dosages did not cause any DNA damage or significant increases in the number of sister chromatid exchanges. The absence of genotoxic effects on lens cells suggests that nanoceria, in the doses and exposures tested in this study, are not deleterious to the eye lens and have the potential for use in studying structural alterations, in developing non-surgical cataract treatments and in investigating other protein folding diseases.

  7. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    SciTech Connect

    Roberts, Joan E. Wielgus, Albert R. Boyes, William K. Andley, Usha Chignell, Colin F.

    2008-04-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C{sub 60}(OH){sub 22-26}] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C{sub 60}(OH){sub 22-26} in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 {mu}M. Exposure to either UVA or visible light in the presence of > 5 {mu}M fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 {mu}M lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein {alpha}-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C{sub 60}(OH){sub 22-26} is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.

  8. Lens and cataract: clastogenic responses in epithelial cells of the organ-cultured rat lens

    SciTech Connect

    Geard, C.R.; Worgul, B.V.

    1987-01-01

    The epithelial cells of the vertebrate lens have an unique character and a probable involvement in cataract formation, which could be initiated by exogenous stimuli. Individual rat lenses were organ-cultured, and the effects of mitomycin C and gamma rays on sister chromatid exchanges (SCE), chromosomal aberrations, and cellular kinetics assessed in cells from the epithelial monolayer. SCE showed about a 5.5-fold increase over the mitomycin C dose range (0, 17, 83, 170 nM), while chromosomal aberrations increased 38-fold. In cells from untreated lenses, SCE were 1600 times more frequent than aberrations and at a level consistent with in vivo assessments in other cell types. Gamma rays (up to 4 Gy) had a greater inhibiting effect on cellular progression, while 17 nM mitomycin C and 1 Gy induced similar clastogenic responses. This first demonstration of such changes in lens epithelial cells expands on the cell types available for monitoring potential mutagen-carcinogens. Additionally chromosomal changes resulting from lens cellular challenge could be the basis of later cytopathological changes in the lens, of which cataract is the primary concern to humans. Potential cataractogens warrant monitoring, and the study outlined may aid in this endeavor, as well as contributing to an understanding of cataract etiology.

  9. Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells.

    PubMed

    Tumminia, S J; Rao, P V; Zigler, J S; Russell, P

    1993-12-08

    Xenobiotic regulatory elements have been identified for enzymes which ameliorate oxidative damage in cells. Zeta (zeta)-crystallin, a taxon-specific enzyme/crystallin shown to be a novel NADPH-dependent quinone reductase, is found in a number of tissues and cell types. This study shows that zeta-crystallin is present in mouse lens epithelium, as well as in the alpha TN4 mouse lens epithelial cell line. To determine whether zeta-crystallin is an inducible quinone reductase, cell cultures were exposed to the xenobiotics, 1,2-naphthoquinone and beta-naphthoflavone. Assays of cellular homogenates showed that quinone reductase activity was stimulated greater than 70% and 90%, respectively, over the control cells. This observed activity was sensitive to dicumarol, a potent inhibitor of quinone reductase activity. 1,2-Naphthoquinone- and beta-naphthoflavone-exposed cells were found to exhibit 1.47- and 1.68-fold increases, respectively, in zeta-crystallin protein concentration. A comparable increase in zeta-crystallin mRNA was indicative of an induction in zeta-crystallin expression in response to naphthalene challenge. Lens epithelial cells were also checked for DT-diaphorase, a well-known cellular protective enzyme which can catalyze the two-electron reduction of quinones. Slot blot analyses indicated that alpha TN4 cells exposed to 1,2-naphthoquinone and beta-naphthoflavone exhibited 2.71- and 6.81-fold increases in DT-diaphorase concentration when compared to the control cells. The data suggest that while DT-diaphorase is most likely responsible for the majority of the observed increase in quinone reductase activity, the zeta-crystallin gene also undergoes activation which is apparently mediated by a xenobiotic-responsive element.

  10. Growth of corneal epithelial cells over in situ therapeutic contact lens after simple limbal epithelial transplantation (SLET).

    PubMed

    Bhalekar, Swapnil; Sangwan, Virender S; Basu, Sayan

    2013-06-27

    An 11-year-old boy underwent simple limbal epithelial transplantation (SLET) from the healthy right eye to his left eye for total limbal stem cell deficiency. One month later, corneal surface epithelialised and whitish plaques overlying the transplants were seen inferiorly. Those plaques were adherent to the surface of the contact lens and underlying corneal surface had smooth elevations. Similar findings were noted in a 23-year man following cyanoacrylate glue application for corneal perforation. On histological and immunohistochemical analysis, cells lining the contact lenses were identified as corneal epithelial cells. These cases illustrate epithelial cell growth on the contact lens and epithelial hyperplasia on corresponding surface of the cornea. Exorbitant proliferation of the epithelial cells may be owing to young age; therefore, early contact lens removal after SLET in young age, can possibly avoid epithelial hyperplasia. This also reiterates the possibility of using contact lens as a scaffold to grow epithelial cells.

  11. Involvement of Egr-1 in lens epithelial cell death induced by selenite.

    PubMed

    Nakajima, T; Belusko, P B; Walkup, R D; Azuma, M; Shearer, T R

    2006-05-01

    Selenite-overdose cataract in young rats may be caused by an initial insult to the lens epithelial cells. Our previous DNA array analysis revealed a significant increase in the expression of mRNA for early growth response protein-1 (Egr-1) in lens epithelial cells after injection of selenite. This suggested that up-regulation of Egr-1 mRNA may be involved in lens epithelial cell death. The purpose of the present experiment was to further clarify the involvement of Egr-1 in lens epithelial cell death induced by selenite. Rat lens epithelial explants were cultured with sodium selenite. Selenite caused epithelial explants to leak LDH into the medium. During LDH leakage, increased expression of mRNA for Egr-1 was observed by RT-PCR. To further test the involvement of Egr-1 in selenite-induced cell death, mouse lens epithelial cell line (alpha-TN4 cells) was treated with antisense oligonucleotide for Egr-1. Antisense oligonucleotide for Egr-1 significantly diminished expression of Egr-1 protein and leakage of LDH. These results suggested that increased activity of Egr-1 may be a factor in lens epithelial cell death induced by selenite.

  12. Effect of intraocular lens design on migration of lens epithelial cells onto the posterior capsule.

    PubMed

    Nagamoto, T; Eguchi, G

    1997-01-01

    To find the conditions that prevent posterior capsule opacification through in vitro analysis of the relationship between intraocular lens (IOL) optic configuration and lens epithelial cell (LEC) migration. Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, Japan. In a preliminary experiment, we measured the size of the capsular bag of rabbits at 8, 16, 20, and 26 weeks of age. The size of a 20-week-old capsular bag was the same size as the capsular bag in senescent Japanese eyes. We isolated the capsular bags in 20- and 8-week-old rabbits. The bags along with a biconvex (BC), convex-plano (CP), or no IOL (C) were cultured, and the eyes were divided into one of six groups (8W-C, 8W-BC, 8W-CP, 20W-C, 20W-BC and 20W-CP), each including six specimens. Two specimens in the 8W-CP group completely blocked LEC migration at the optic edge. All specimens in the 20W-CP group and one in the 20W-BC group showed cell aggregation along the optic edge. None of the other specimens in the BC and C groups blocked migration or showed cell aggregation. In the rabbit-model study, the convex-plano lens was superior to the biconvex lens in inhibiting migration of LECs. A firm contact between the IOL and the posterior capsule blocked the migration.

  13. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  14. A gradient of matrix-bound FGF-2 and perlecan is available to lens epithelial cells.

    PubMed

    Wu, Weiju; Tholozan, Frederique M; Goldberg, Martin W; Bowen, Leon; Wu, Junjie; Quinlan, Roy A

    2014-03-01

    Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/μm2; equator: 8.2 ± 1.9 particles/μm2; posterior pole: 4 ± 0.9 particles/μm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/μm2; equator: 5 ± 2 particles/μm2; posterior pole: 1.9 ± 0.7 particles/μm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface.

  15. Regulation of potassium transport in human lens epithelial cells.

    PubMed

    Lauf, Peter K; Warwar, Ronald; Brown, Thomas L; Adragna, Norma C

    2006-01-01

    The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K-Cl cotransport (KCC). At 0.3-0.5 mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished approximately 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 microM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.

  16. KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts.

    PubMed

    Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C; Warwar, Ronald; Brown, Thomas L; Lauf, Peter K

    2006-11-01

    We recently reported potassium-chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium-chloride cotransport (KCC) isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT-PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.

  17. Taurine Protects Lens Epithelial Cells Against Ultraviolet B-Induced Apoptosis.

    PubMed

    Dayang, Wu; Dongbo, Pang

    2017-10-01

    The massive uptake of compatible osmolytes is a self-protective response shared by lens exposed to hypertonic stress and ultraviolet stress. This study aimed to investigate the protective effects of taurine against ultraviolet B-induced cytotoxicity in the lens epithelial cells. Real-time PCR was used to measure osmolytes transport. Radioimmunoassay was used to measure osmolytes uptake. Cell counting kit-8 assays were used to measure cellular viability. Flow cytometry analysis was used to measure apoptosis level. Compared with normotonic stress, hypertonic stress-induced osmolytes uptake into the lens epithelial cells such as betaine, myoinositol and taurine. UVB exposure increased osmolytes transporter mRNA expression together with osmolytes uptake. Moreover, taurine suppressed UVB-induced cell apoptosis in the lens epithelial cells significantly. The effect of compatible osmolyte taurine on cell survival rate may play an important role in cell resistance and adaption to UVB exposure.

  18. Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite.

    PubMed

    Belusko, P B; Nakajima, T; Azuma, M; Shearer, T R

    2003-10-13

    An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.

  19. Cell-autonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis.

    PubMed

    Rivera, Charlene; Yamben, Idella F; Shatadal, Shalini; Waldof, Malinda; Robinson, Michael L; Griep, Anne E

    2009-09-01

    Cell polarity and adhesion are thought to be key determinants in organismal development. In Drosophila, discs large (dlg) has emerged as an important regulator of epithelial cell proliferation, adhesion, and polarity. Herein, we investigated the role of the mouse homolog of dlg (Dlg-1) in the development of the mouse ocular lens. Tissue-specific ablation of Dlg-1 throughout the lens early in lens development led to an expansion and disorganization of the epithelium that correlated with changes in the distribution of adhesion and polarity factors. In the fiber cells, differentiation defects were observed. These included alterations in cell structure and the disposition of cell adhesion/cytoskeletal factors, delay in denucleation, and reduced levels of alpha-catenin, pERK1/2, and MIP26. These fiber cell defects were recapitulated when Dlg-1 was disrupted only in fiber cells. These results suggest that Dlg-1 acts in a cell autonomous manner to regulate epithelial cell structure and fiber cell differentiation.

  20. Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    PubMed Central

    Goralska, Malgorzata; Fleisher, Lloyd N.; McGahan, M. Christine

    2017-01-01

    Purpose In humans, vitrectomy is associated with development of nuclear cataracts. Iron catalyzes free radical formation causing oxidative damage, which is implicated in cataract formation. This study was designed to determine if vitreous humor, which can initiate differentiation of lens epithelial cells, would have an effect on iron-handling proteins. Methods Cultured canine lens epithelial cells were treated with collected canine vitreous humor. Lysates of treated and control cells were separated by SDS-PAGE. Ferritin H- and L-chains, transferrin receptor 1, and aquaporin 0 were immunodetected and quantitated with specific antibodies. Morphologic changes in treated cells were assessed. Results Treatment of lens epithelial cells with a 33% (vol/vol) solution of vitreous humor changed the morphology of lens cells and induced expression of aquaporin 0, a marker of fiber cell differentiation that was undetectable in control cells. Treatment did not modify the size of iron-handling proteins but significantly increased content of ferritin from 2.9- to 8.8-fold over control and decreased levels of transferrin receptor by 37% to 59%. Conclusions Vitreous humor may significantly limit iron uptake by transferrin/transferrin receptor pathway, and by increasing ferritin levels could profoundly increase the iron-storage capacity of ferritin in lens cells. Vitreous humor may play a significant protective role against iron-catalyzed oxidative damage of lens epithelial cells and therefore in the formation of cataracts. PMID:28245299

  1. Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism

    PubMed Central

    Dawes, LJ; Sugiyama, Y; Lovicu, FJ; Harris, C; Shelley, EJ; McAvoy, JW

    2013-01-01

    How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behaviour of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes. PMID:24211762

  2. Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells.

    PubMed

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.

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

    PubMed Central

    Raghavan, Cibin T.; Nagaraj, Ram H.

    2016-01-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis. PMID:27263094

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

    PubMed

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

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

  5. The leukocyte chemotactic receptor FPR1 is functionally expressed on human lens epithelial cells.

    PubMed

    Schneider, Erich H; Weaver, Joseph D; Gaur, Sonia S; Tripathi, Brajendra K; Jesaitis, Algirdas J; Zelenka, Peggy S; Gao, Ji-Liang; Murphy, Philip M

    2012-11-23

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

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

    PubMed

    Kidd, G L; Reddan, J R; Russell, P

    1994-04-01

    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.

  7. In vitro inhibition of lens epithelial cell growth by continuous wave Nd:YAG laser

    SciTech Connect

    Miyake, K.; Iwata, S.; Ando, F.; Daikuzono, N.; Federman, J.L.

    1989-04-01

    Bovine lens epithelial cells were suspended in MEM medium and subjected to continuous wave, low power, pulsed neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation. The temperature of each suspension was maintained at 36 degrees C. Laser applications ranged from 1 to 10 watts and from 100 to 2000 seconds, but the total dose to each of the epithelial cell suspension was 2000 J. Six to thirty-nine percent of the cells were dead immediately after irradiation. Surviving cells, cultured for 15 days, showed decreased attachment and failed to grow. These preliminary results suggest that the Nd:YAG laser may be used during cataract surgery to prevent subsequent lens epithelial cell proliferation and the resulting vision reduction and glare.

  8. Lens epithelial cell apoptosis and intracellular Ca2+ increase in the presence of xanthurenic acid

    PubMed Central

    Malina, Halina; Richter, Christoph; Frueh, Beatrice; Hess, Otto M

    2002-01-01

    Background Xanthurenic acid is an endogenous product of tryptophan degradation by indoleamine 2,3-dioxygenase (IDO). We have previously reported that IDO is present in mammalian lenses, and xanthurenic acid is accumulated in the lenses with aging. Here, we studied the involvement of xanthurenic acid in the human lens epithelial cell physiology. Methods Human lens epithelial cells primary cultures were used. Control cells, and cells in the presence of xanthurenic acid grow in the dark. Western blot analysis and immunofluorescence studies were performed. Results In the presence of xanthurenic acid human lens epithelial cells undergo apoptosis-like cell death. In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 μM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown. In the same condition caspase-3 activation, and DNA fragmentation was observed. At low (5 to 10 μM) of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release. At higher concentrations xanthurenic acid (20 μM and 40 μM) damaged mitochondria were observed in the perinuclear region, and nuclear DNA cleavage was observed. We observed an induction of calpain Lp 82 and an increase of free Ca2+ in the cells in a xanthurenic acid concentration-dependent manner. Conclusions The results show that xanthurenic acid accumulation in human lens epithelial cells disturbs the normal cell physiology and leads to a cascade of pathological events. Xanthurenic acid induces calpain Lp82 and caspases in the cells growing in the dark and can be involved in senile cataract development. PMID:11934353

  9. Conservation of mouse alpha A-crystallin promoter activity in chicken lens epithelial cells.

    PubMed

    Donovan, D M; Sax, C M; Klement, J F; Li, X; Chepelinsky, A B; Piatigorsky, J

    1992-10-01

    Previous transfection experiments have shown that 162 base pairs (bp) of the 5' flanking sequence of the chicken alpha A-crystallin gene are required for promoter activity in primary chicken lens epithelial cells (PLE), while only 111 bp of the 5' flanking sequence are needed for activity of the mouse alpha A-crystallin promoter in transfected chicken PLE cells or in a SV40 T-antigen-transformed transfected mouse lens epithelial cell line (alpha TN4-1). The effect of site-directed mutations covering positions -111 to -34 of the mouse alpha A-crystallin promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was compared in transfected chicken PLE cells and mouse alpha TN4-1 cells; selected mutations were also examined in a nontransformed rabbit lens epithelial cell line (N/N1003A). In general, the same mutations reduced promoter activity in the transfected lens cells from all three species, although differences were noted. The mutations severely affected regions -111/-106 and -69/-40 regions in all the transfected cells examined; by contrast, mutations at positions -105/-99 and -87/-70 had a somewhat greater effect in the chicken PLE than the mouse alpha TN4-1 cells, while mutations of the -93/-88 sequence reduced expression in the alpha TN4-1 but not the PLE cells. A partial cDNA with sequence similarity to alpha A-CRYPB1 of the mouse has been isolated from a chicken lens library; mouse alpha A-CRYBP1 is a putative transcription factor which binds to the -66/-55 sequence of the mouse alpha A-crystallin promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. β-Catenin/CBP–Dependent Signaling Regulates TGF-β–Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells

    PubMed Central

    Taiyab, Aftab; Korol, Anna; Deschamps, Paula A.; West-Mays, Judith A.

    2016-01-01

    Purpose Transforming growth factor-β–induced epithelial–mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-β–induced PCO have not been fully characterized. Here, we focus on examining the role of β-catenin/cyclic AMP response element–binding protein (CREB)-binding protein (CBP) and β-catenin/T-cell factor (TCF)-dependent signaling in regulating cytoskeletal dynamics during TGF-β–induced EMT in lens epithelial explants. Methods Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-β2 in the presence or absence of the β-catenin/CBP interaction inhibitor, ICG-001, or the β-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed. Results An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-β, and colocalized with F-actin filaments. Inhibition of β-catenin/CBP interactions, but not β-catenin/TCF interactions, led to a decrease in TGF-β–induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, α-smooth muscle actin (α-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of β-catenin/CBP–dependent signaling also prevented TGF-β–induced downregulation of epithelial cadherin (E-cadherin) in lens explants. Conclusions We show that β-catenin/CBP–dependent signaling regulates fascin, MMP9, and α-SMA expression during TGF-β–induced EMT. We demonstrate that β-catenin/CBP–dependent signaling is crucial for TGF-β–induced EMT in the lens. PMID:27787561

  11. [TAK1 promotes epithelial-mesenchymal transition of lens epithelial cells].

    PubMed

    Dong, N; Tang, X; Yuan, X Y; Song, H; Li, J

    2016-04-11

    Transforming growth factor-β-activated kinase-1 (TAK1) is thought to play a key role in the initiation of Smad-independent TGF-β signaling. This study investigated the role of TAK1 in the epithelial-mesenchymal transition (EMT) lens epithelial cells. TAK1 was overexpressed in the HLE B-3 cell line by transfecting TAK1-pcDNA3 and TAK1-binding protein 1 (TAB1)-pcDNA3 plasmids. The expression levels of TAK1, phospho-TAK1, E-cadherin, and fibronectin were detected by Western blot analysis and immunocytofluorescence to analyze the effects of overexpression. The levels of α-SMA and type I collagen were analyzed by real-time PCR. Quantitative data were analyzed by Student's t test or one-way analysis of variance (ANOVA) (multiple comparisons using LSD test). Western blot analysis showed in the TAK1-pcDNA3 plasmids group, expression of TAK1 proteins (1.00±0.03) with a maximum upregulation of approximately 80% at 24 h than it was in the control group (0.19±0.09)(t=8.02, P< 0.01); Western blot analysis showed in the TAB1-pcDNA3 plasmids group, expression of TAB1 proteins (1.00±0.02) with a maximum upregulation of approximately 78% at 24 h than it was in the control group (0.22±0.08)(t=7.63, P<0.01). The levels of E-cadherin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (1.00±0.02, 0.12±0.03, 0.98±0.09, 0.92±0.08;F=31.03, P<0.01). The levels of fibronectin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (0.11±0.03, 1.00±0.05, 0.16±0.04, 0.21±0.05;F=35.12, P<0.01). Overexpression of TAK1 with TAB1 resulted in upregulated expression of fibronectin, and downregulated expression of E-cadherin. The expression of E-cadherin was increased and the expression of fibronectin was decreased by TAK1 siRNA and TAK1 chemical inhibitors in the presence of TGF-β2. These data

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

    PubMed Central

    Schneider, Erich H.; Weaver, Joseph D.; Gaur, Sonia S.; Tripathi, Brajendra K.; Jesaitis, Algirdas J.; Zelenka, Peggy S.; Gao, Ji-Liang; Murphy, Philip M.

    2012-01-01

    Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1−/− mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ∼2500 specific binding sites on FHL-124 cells (KD ∼ 0.5 nm) versus ∼40,000 sites on neutrophils (KD = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca2+ flux in FHL 124 cells, consistent with classic Gi-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice. PMID:23012360

  13. Mitochondrial Oxygen Metabolism in Primary Human Lens Epithelial Cells: Association with Age, Diabetes and Glaucoma

    PubMed Central

    Kubota, M; Shui, YB; Liu, M; Bai, F; Huang, AJ; Ma, N; Beebe, DC; Siegfried, CJ

    2016-01-01

    Purpose The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. Methods Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U-test and multivariate regression analysis. Results Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21 ± 1.037 vs. 2.86 ± 1.383 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02 ± 0.911 vs. 2.79 ± 1.332 fmol/min/cell; p<0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27 ± 1.19 vs. 2.83 ± 1.286 fmol/min/cell; p<0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p<0.05), diabetic patients (p<0.01), and glaucoma patients (p<0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR. Conclusions The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma

  14. Mitochondrial oxygen metabolism in primary human lens epithelial cells: Association with age, diabetes and glaucoma.

    PubMed

    Kubota, M; Shui, Y B; Liu, M; Bai, F; Huang, A J; Ma, N; Beebe, D C; Siegfried, C J

    2016-08-01

    The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis. Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p<0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p<0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p<0.05), diabetic patients (p<0.01), and glaucoma patients (p<0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR. The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes

  15. Presence of melanopsin in human crystalline lens epithelial cells and its role in melatonin synthesis.

    PubMed

    Alkozi, Hanan Awad; Wang, Xiaoyu; Perez de Lara, Maria J; Pintor, Jesus

    2017-01-01

    Melanopsin is a non-image forming photoreceptor known to be present in the retina and it is considered to have light regulated tasks among other functions. In the present work, melanopsin presence in human lens epithelial cells as well as in human lens tissue is described for the first time. Moreover, studying the concentration of melatonin and its synthesising enzyme AANAT proved a clear link between melanopsin activation and the suppression of melatonin synthesis. Melanopsin sensitivity to specific wavelength (465-480 nm, blue) was confirmed after making temporal studies incubating lens epithelial cells under light, red, green, blue and total darkness for 2, 4, 8, 12 h and analysing the concentration of both melatonin and its synthesising enzyme AANAT, discovering that melatonin levels after submitting cells to total darkness are significantly higher to ones submitted to white or specifically blue light (***p < 0.001, n = 6). The involvement of melanopsin in the regulation of melatonin was also determined by using a specific inhibitor AA92593 and by inhibiting melanopsin-induced phospholipase C activation. Under this situation neither AANAT nor melatonin levels changed under light conditions (n = 4, ***p < 0.001). The discovery of melanopsin in the lens opens the possibility of regulating melatonin synthesis with the corresponding implication as an antioxidant substance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Lycium barbarum Polysaccharides Protect Human Lens Epithelial Cells against Oxidative Stress–Induced Apoptosis and Senescence

    PubMed Central

    Wen, Yuechun; Liu, Lian; Guo, Xiaoling; Hou, Guanghui; Wang, Guifang; Zhong, Jingxiang

    2014-01-01

    Objectives We aimed to investigate the protective effect of Lycium barbarum polysaccharides (LBPs) against oxidative stress–induced apoptosis and senescence in human lens epithelial cells. Methods To study apoptosis, SRA01/04 cells, a human lens epithelial cell lines, were exposed to 200 µM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with LBPs. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, intracellular reactive oxygen species (ROS), and the loss of mitochondria membrane potential (Δψm) were detected by flow cytometric analyses. Expression levels of Bcl-2 and Bax proteins were measured by western blot analysis. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were quantized using commercial enzymatic kits according to the manufacturer's instructions. To study senescence, SRA01/04 cells were pre-incubated with LBPs and all cells were then exposed to 100 µM H2O2 for 96 h. Cellular senescence was assessed by morphologic examination and senescence-associated β-galactosidase (SA-β-gal) staining. Results LBPs significantly reduced H2O2-induced cell apoptosis, the generation of ROS, the loss of Δψm, and the levels of MDA. LBPs also inhibited H2O2-induced downregulated Bcl-2 and upregulated Bax proteins and increased the levels of SOD and GSH enzyme activity. Moreover, LBPs significantly attenuated H2O2-induced cellular senescence. Conclusions These findings suggested that LBPs protect human lens epithelial cells from H2O2-induced apoptosis by modulating the generation of ROS, loss of Δψm, Bcl-2 family, and antioxidant enzyme activity and attenuating cellular senescence. PMID:25333784

  17. Mitomycin C-induced cell death in mouse lens epithelial cells.

    PubMed

    Park, Hyun-Kyung; Lee, Kwang-Won; Choi, Jun-Sub; Joo, Choun-Ki

    2002-01-01

    The mode of action of mitomycin C (MMC)-induced cell death in lens epithelia was investigated using cell culture system. The cytotoxicity of MMC on alpha-TN4 mouse lens epithelial cells (LECs) was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay. To determine the type of cell death induced by MMC, we stained LECs with acridine orange and ethidium bromide, and performed TUNEL assay and electron microscopic examination. The activation of MMC was also investigated with N-acetly-L-cystein (NAC), a free radical scavenger, and dicumarol, an inhibitor of DT-diaphorase, to analyze one- and two-electron reduction pathways involved in the activation of MMC. MTT assay showed that 400 microg/ml of MMC decreased the cell viability of the control cells to 45%, and the cytotoxicity of MMC on alpha-TN4 cells was increased in a dose-dependent manner. Based on the results obtained from acridine orange and ethidium bromide staining, the TUNEL assay, and transmission electron microscopic examination, we confirmed that MMC-induced cell death, at 400 microg/ml of MMC, occurs with necrosis as well as apoptotis. The treatment of cells with 2 mM NAC for 1 h or 20 microM dicumarol for 30 min, prior to 5 min of MMC (400 microg/ml) treatment, restored the growth suppression to 76 and 80% of the control level, respectively. In addition, the cotreatment of cells with NAC and dicumarol restored cell viability to 90% of the control level. The cellular death in MMC-treated LECs showed the involvement of oxidative stress in this apoptosis accounting for 22% of the observed cell death at 400 microg/ml of MMC.

  18. Presence of beta-citryl-L-glutamic acid in the lens: its possible role in the differentiation of lens epithelial cells into fiber cells.

    PubMed

    Tsumori, M; Asakura, M; Narahara, M; Ogawa, T; Nakae, M; Nakagawa, S; Kawai, Y; Morino, H; Hama, T; Miyake, M

    1995-10-01

    The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG in the bovine eye was determined. About 95% of total beta-CG content in the whole eye was localized in the lens. However, the distribution of beta-CG in the eye varied depending on species. beta-CG was exclusively localized in the lens in the eyes of fish and mammals, but distributed in both lens and retina in frogs. The molecule was localized in the retina rather than the lens in the chicken eye, although the concentrations was extremely low compared to those in the mammalian, amphibian and fish eyes. It was found that beta-CG is present ubiquitously in the lens or retina in various species. The distribution of beta-CG in the bovine lens was determined in the three cortex regions and nucleus. beta-CG was present at the highest concentration in the equatorial cortex, at a moderate concentration in the posterior and anterior cortex, and at the lowest concentration in the nucleus. Similar distribution patterns were also found in the rabbit and rat lens. When embryonic chick lens epithelial cells were cultured in the presence of fetal calf serum, the cells elongated, differentiated into fiber cells and formed lentoid bodies. The cells of lentoid bodies were stained strongly by the anti-beta-CG antibody, while cells around the structures were not. In addition, the beta-CG content in the lenses from the galactose cataractous rat decreased to about 20-30% of that in the normal lens. These findings suggest that beta-CG may play a role in the differentiation of epithelial cells into fiber cells.

  19. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    PubMed Central

    Youn, Hyun-Yi; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. Results The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusions The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the

  20. Inhibition of lens epithelial cell migration at the intraocular lens optic edge: role of capsule bending and contact pressure.

    PubMed

    Nagamoto, Toshiyuki; Fujiwara, Takaaki

    2003-08-01

    To evaluate the inhibitory effect of a sharp intraocular lens (IOL) optic edge, a sharp capsule bend, and contact pressure between the optic edge and posterior capsule on lens epithelial cell (LEC) migration. Department of Ophthalmology, Kyorin University, Tokyo, Japan. This in vitro laboratory study evaluated a tumble-polished convex-plano IOL (CP group), an AcrySof IOL (Alcon) with a sharp edge (AS group), a new IOL with a round ridge (RR group), and a new IOL with a sharp ridge (SR group). The 2 new IOLs have high ridges and high angled loops that create firm contact between the ridge and posterior capsule. After sham cataract surgery, an IOL and a capsular tension ring (CTR) were implanted in the capsular bag of rabbit eyes. The extracted capsular bags containing the CTR and IOL were cultured. The inhibitory effect of each IOL on cell migration was analyzed. Furthermore, LEC migration on the posterior capsule was compared in culture between capsules having a sharp right angle and those with gradually curving bends. The inhibitory effect on cell migration was statistically greatest in the SR group followed by the RR, AS, and CP groups. A sharp capsule bend did not inhibit cell migration. The results suggest that inhibition of cell migration at the optic edge is regulated by the degree of contact pressure between the optic edge and posterior capsule. A sharp capsule bend might indicate strong contact but does not in itself inhibit cell migration.

  1. Ferulic Acid Suppresses Amyloid β Production in the Human Lens Epithelial Cell Stimulated with Hydrogen Peroxide

    PubMed Central

    Nagai, Noriaki; Kotani, Sachiyo; Mano, Yu; Ueno, Akina; Ito, Yoshimasa; Kitaba, Toshio; Takata, Takumi

    2017-01-01

    It is well known that oxidative stresses induce the production of amyloid β (Aβ) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aβ levels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aβ peptides (Aβ1-40, Aβ1-42, and Aβ1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aβ production (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2 stimulation augmented gene expression of the proteins related to Aβ production, resulting in the production of three types of Aβ peptides. Treatment with 0.1 μM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aβ peptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells. PMID:28409157

  2. Establishment of a lens epithelial cell line from a canine mature cataract.

    PubMed

    Kanemaki, Nobuyuki; Saito, Miyoko; Onda, Ken; Maruo, Takuya; Ogihara, Kikumi; Naya, Yuko; Morishita, Taiki; Ochiai, Hideharu

    2012-01-01

    The aim of this study was to establish a lens epithelial cell (LEC) line originated from a cataract of a dog. An anterior capsulorhexis specimen from a dog naturally developing mature cataracts was obtained prior to routine phacoemulsification cataract extraction. The primary lens epithelial cells were transfected with expression plasmid DNA encoding the large T antigen of replication origin-defective simian virus 40 (SV40), and then a colony was cloned using a glass cylinder. The primary cells stopped proliferation in three passages, while the transfected cells remained proliferative. Functional analysis of Na-dependent vitamin C transporter (SVCT) indicated that the Km value toward ascorbic acid (vitamin C) was 19.9 ± 2.8 µM, and RT-PCR analysis showed that SVCT2 was observed in this cell line while SVCT1 was not, which is one of the characteristics of LECs. Western blot analysis and cytoimmunochemistry indicated immortalized cells produced a protein with a molecular mass of 25 kDa, which reacted with an antibody to αB-crystallin within the whole cytosol. The cloned cell line, termed cdLEC, grew well and could be propagated over 250 times by basically splitting at 1:20. These results indicate that cdLEC may also provide a useful in vitro system for the study of the pathophysiology of cataract.

  3. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. While in the cortex and nucleus radiation interacts with proteins, experimental results from cultured lenses and lens epithelial cells demonstrate mutagenic and cytotoxic effects in the epithelium. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the radiation's relative biological effectiveness (RBE), because cosmic rays differ significantly from X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiations. Irradiations were performed either with 300 kV X-rays or at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. For strand break measurements hydroxyapatite chromatography of alka-line unwound DNA (overall strand breaks) and non-denaturing filter elution technique (double strand breaks) were applied. Experiments showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV/μm more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of

  4. BAX gene over-expression via nucleofection to induce apoptosis in human lens epithelial cells.

    PubMed

    Fang, Yanwen; Mo, Xiaofen; Luo, Yi; Lu, Yi

    2012-09-01

    Despite significant advances in cataract surgery techniques, posterior capsule opacification (PCO) remains a common complication. In PCO, remaining epithelial cells cloud the lens capsule and impair postoperative vision. This in vitro study was designed to investigate the potential of a gene-based approach, specifically over-expression of the proapoptotic BAX gene, to prevent PCO. Human lens epithelial cells (HLECs) were transfected by nucleofection with a plasmid encoding a fusion protein of green fluorescent protein and human BAX. The expression levels of BAX and its antiapoptotic counterpart BCL2 were determined by realtime reverse transcription polymerase chain reaction, Western blotting and immunofluorescence. BAX over-expression-induced cell death was analyzed by fluorescence-activated cell sorting using the Annexin V antibody. Fluorescence microscopy and transmission electron microscopy were used to assess changes in morphology and ultrastructure. Differential expression of the downstream apoptosis-related factor, caspase 3, was detected by Western blotting. Nucleofection efficiency was high (nearly 80%). BAX-transfected HLECs showed remarkably enhanced BAX gene expression and BAX:BCL2 ratio, but relatively little change in endogenous BCL2 expression. BAX over-expression also led to significant cytotoxicity, induction of apoptosis-related characteristics and activation of caspase 3. In conclusion, our results indicate that BAX gene over-expression can trigger cell death in HLECs via an apoptotic pathway. Thus, BAX may be a promising candidate for human gene therapy to treat PCO.

  5. Hypericin-Mediated Photooxidative Damage of α-crystallin in Human Lens Epithelial Cells

    PubMed Central

    Ehrenshaft, Marilyn; Roberts, Joan E.; Mason, Ronald P.

    2013-01-01

    St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used and appears to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and UVA range, is considered to be one of the bioactive ingredients, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. Alpha-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens, and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescent confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400 nm provides incomplete protection against α-crystallin modifications and NFK accumulation, suggesting that even by wearing UV blocking sunglasses, routine users of St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage. PMID:23453985

  6. Hypericin-mediated photooxidative damage of α-crystallin in human lens epithelial cells.

    PubMed

    Ehrenshaft, Marilyn; Roberts, Joan E; Mason, Ronald P

    2013-07-01

    St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used for and seems to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and the UVA range, is considered to be one of the bioactive ingredients of St. John's wort, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. α-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescence confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400nm provides incomplete protection against α-crystallin modification and NFK accumulation, suggesting that even by wearing UV-blocking sunglasses, routine users of St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage.

  7. Ablation of lens epithelial cells with a laser photolysis system: Histopathology, ultrastructure, and immunochemistry

    PubMed Central

    Mamalis, Nick; Grossniklaus, Hans E.; Waring, George O.; Werner, Liliana; Brubaker, Jacob; Davis, Don; Espandar, Ladan; Walker, Rudolf; Thyzel, Reinhardt

    2010-01-01

    PURPOSE To evaluate efficacy of a neodymium:YAG (Nd:YAG) laser photolysis system in removing lens epithelial cells (LECs) and characterize the effect of the laser on laminin and fibronectin involved in LEC adhesion and migration. METHODS Cadaver eyes were evaluated using the Miyake technique. The lenses were removed with phacoemulsification. The modified Nd:YAG laser was used to clean the LECs from the capsule. Only the fornix was cleaned in some eyes and the anterior subcapsular area in other eyes. Some areas were not treated and acted as controls. Standard irrigation/aspiration (I/A) removal of LECs was performed in additional eyes. The eyes were analyzed using light microscopy and immunohistochemical staining. RESULTS Histopathologic evaluation showed that the laser removed the LECs from the anterior lens capsule and from the fornix. Immunohistochemical staining showed fibronectin and laminin staining in the untreated areas that was absent in the treated areas. Standard I/A removal of the LECs showed absence of cells but persistent laminin and fibronectin. Electron microscopy showed epithelial cells in untreated areas with an absence of the LECs and debris in treated areas. CONCLUSIONS The laser photolysis system removed LECs from the anterior lens capsule and capsule fornix. Along with the cells, laminin, fibronectin, and cell debris remained in the untreated areas but were removed by the treatment. This treatment may be useful in preventing posterior capsule opacification. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes. PMID:20494774

  8. Growth and differentiation of human lens epithelial cells in vitro on matrix

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

    2000-01-01

    PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

  9. Growth and differentiation of human lens epithelial cells in vitro on matrix

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

    2000-01-01

    PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

  10. Ganglioside GM3 participates in the TGF-β1-induced epithelial-mesenchymal transition of human lens epithelial cells.

    PubMed

    Kim, Seok-Jo; Chung, Tae-Wook; Choi, Hee-Jung; Kwak, Choong-Hwan; Song, Kwon-Ho; Suh, Seok-Jong; Kwon, Kyung-Min; Chang, Young-Chae; Park, Young-Guk; Chang, Hyeun Wook; Kim, Kyoung-Sook; Kim, Cheorl-Ho; Lee, Young-Choon

    2013-01-01

    TGF-β (transforming growth factor-β)-induced EMT (epithelial-mesenchymal transition) induces the proliferation and migration of the HLE (human lens epithelial) cells. Ganglioside GM3, simple sialic-acid-containing glycosphingolipids on mammalian cell membranes, regulates various pathological phenomena such as insulin resistance and tumour progression. However, the relationship between ganglioside GM3 and TGF-β-induced EMT in the HLE B-3 cells is poorly understood. In the present study we demonstrated that ganglioside GM3 was involved in TGF-β1-induced EMT in HLE B-3 cells. Our results indicated that the expression of ganglioside GM3 and GM3 synthase mRNA were significantly increased in TGF-β1-induced HLE B-3 cells. Reporter gene analysis also demonstrated that transcriptional activation of the GM3 synthase gene was regulated by Sp1 (specificity protein 1) in HLE B-3 cells upon TGF-β1 stimulation. Interestingly, the inhibition of ganglioside GM3 expression by d-PDMP [d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol] and GM3 synthase shRNA (short hairpin RNA) resulted significantly in the suppression of cell migration and EMT-related signalling in HLE B-3 cells stimulated by TGF-β. Furthermore, exogenous treatment of ganglioside GM3 rescued the expression of EMT molecules and cell migration suppressed by the depletion of ganglioside GM3 in TGF-β1-induced HLE B-3 cells. We also found that ganglioside GM3 interacted with TGFβRs (TGF-β receptors) in TGF-β1-induced HLE B-3 cells. Taken together, these results suggest that ganglioside GM3 induced by TGF-β1 regulates EMT by potential interaction with TGFβRs.

  11. Curcumin inhibits proliferation of human lens epithelial cells: a proteomic analysis*

    PubMed Central

    Hu, Yan-hong; Huang, Xiu-rong; Qi, Ming-xin; Hou, Bu-yuan

    2012-01-01

    Objective: The incidence of after-cataracts [also known as posterior capsular opacification (PCO)] is between 30% and 50% three years following cataract surgery. Suppressing the proliferation of lens epithelial cells (LECs) is a primary goal in preventing PCO. Here, we investigated the proteomic regulation of the inhibitory effects of curcumin (Cur) on the proliferation of human lens epithelial B3 (HLE-B3) cells. Methods: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 20 mg/L Cur in a CO2 incubator for 24 h. Results: We found that the absorbance (A) value of rhbFGF group was significantly higher than the A value of the control group. Furthermore, the A value of the Cur group was significantly lower compared to the rhbFGF group, with an inhibition of 53.7%. Five different protein spots were obtained from proliferative HLE-B3 cells induced by rhbFGF. Eight different protein spots were obtained in HLE-B3 cells incubated with Cur. There were the common variational protein spots at mass/charge (m/z) ratios of 8 093 and 13 767 between rhbFGF group and control group as well as between the Cur group and rhbFGF group. Conclusions: These results show that Cur effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. The protein spots at m/z of 8 093 and 13 767 may be the targets of Cur-induced inhibition of HLE-B3 cell proliferation. Cur may be a reliable and effective drug for prevention and treatment of polymerase chain reaction (PCR). PMID:22556179

  12. Mechanisms by which ascorbic acid increases ferritin levels in cultured lens epithelial cells.

    PubMed

    Goralska, M; Harned, J; Grimes, A M; Fleisher, L N; McGahan, M C

    1997-03-01

    A previous study demonstrated that ascorbic acid increased the concentration of the iron storage protein, ferritin. In cultured lens epithelial cells. The current study was designed to determine the mechanism by which ascorbic acid exerts this effect. Ascorbic acid increased both ferritin mRNA levels (by about 30%) and translation of ferritin (de novo synthesis was increased up to 15-fold) within 6 hr. Cycloheximide completely abolished the ability of ascorbic acid to increase ferritin levels, whereas actinomycin D only decreased it by about 30%. Therefore, the ascorbic-acid induced increase in ferritin concentration is due mainly to an increase in ferritin synthesis at the translational levels. This is a novel role for ascorbic acid. Addition of iron with ascorbic acid further increased de novo synthesis of ferritin, but this additive effect was only noted at a later time point (20 hr). Factors which decrease ferritin mRNA translation, such as the reducing agent dithiothreitol or the iron chelator desferrioxamine, reduced the ascorbic acid effect on de novo ferritin synthesis. The effects of ascorbic acid on ferritin mRNA levels may be mediated by its oxidation product, H2O2, since, like ascorbic acid, H2O2 increased ferritin mRNA levels by 30%. However, in contrast to the ascorbic acid-induced increase in translation of ferritin, H2O2 substantially decreased de novo ferritin synthesis. This effect of H2O2 could have physiological significance in eyes where concentrations of H2O2 in the aqueous humor are elevated. High levels of H2O2 could decrease the concentration of ferritin within the lens. Since ferritin sequesters iron and has been shown to decrease oxidative damage by limiting the availability of iron to catalyse free radical reactions, H2O2-induced reduction in ferritin concentration in the lens could have deleterious effects. The ability of ascorbic acid to increase ferritin concentration in lens epithelial cells could provide an additional protective

  13. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

    PubMed

    Baumstark-Khan, C; Heilmann, J; Rink, H

    2003-01-01

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of

  14. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ -1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non

  15. CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.

    PubMed

    Zhang, W; Liu, Z; Bao, X; Qin, Y; Taylor, A; Shang, F; Wu, M

    2015-01-01

    Protein quality control (PQC) systems, including molecular chaperones and ubiquitin-proteasome pathway (UPP), plays an important role in maintaining intracellular protein homeostasis. Carboxyl terminus of Hsc70- interacting protein (CHIP) links the chaperone and UPPs, thus contributing to the repair or removal of damaged proteins. Over-expression of CHIP had previously been used to protect cells from environmental stress. In order to gain a more physiologic mechanism of the advantage conferred by CHIP, we induced a CHIP knockdown and monitored the ability of cells to cope with environmental stress. To knockdown CHIP, the human lens epithelial cell line HLE B3 was transfected with lentiviral particles that encode a CHIP short hairpin RNA (shRNA) or negative control lentiviral particles. Stable CHIP-knock down cells (KD) and negative control cells (NC) were selected with puromycin. After exposure to heat shock stress, there was no change observed in the expression of Hsp90. In contrast, Hsp70 levels increased significantly in NC cells but less so in KD cells. Hsp27 levels also increased after heat shock, but only in NC cells. Protein ubiquitination was reduced when CHIP was knocked down. CHIP knockdown reduced the ability to clear aggregation proteins. When same levels of aggregation-prone RFP-mutant crystallin fusion protein, RFP/V76D-γD, was expressed, there was ~9- fold more aggregates in KD cells as compared to that observed in NC cells. Furthermore, KD cells were more sensitive to toxicity of amino acid analog canavanine as compared to NC cells. Together, these data indicate that CHIP is required for PQC and that CHIP knockdown diminished cellular PQC capacity in lens cells.

  16. The effect of ascorbic acid and ferric ammonium citrate on iron uptake and storage in lens epithelial cells.

    PubMed

    Goralska, M; Harned, J; Fleisher, L N; McGahan, M C

    1998-06-01

    Ferritin is the major intracellular iron storage protein which has been shown to protect cells against oxidative damage. Recent reports that an inherited abnormality in human ferritin synthesis is associated with early bilateral cataracts underscore the importance of understanding ferritin synthesis and iron storage in lens epithelial cells. We previously demonstrated that ascorbic acid greatly increases de novo synthesis of ferritin in lens epithelial cells. The objectives of the present study were to determine: (1) the effects of ascorbic acid and ferric ammonium citrate on iron uptake by canine lens epithelial cells from iron bound to transferrin and from ferric chloride and (2) the incorporation of this element into ferritin. Iron uptake by lens epithelial cells from 59ferric chloride was 20 times higher than from 59iron-transferrin and iron deposition into ferritin was 8-fold higher when 59ferric chloride was the source. Ascorbic acid had a stimulatory effect on iron uptake from transferrin and on incorporation of this element into ferritin. The ascorbic acid-induced increase of iron uptake required de novo protein synthesis but not specifically de novo ferritin biosynthesis. Although ferritin is not directly involved in iron uptake, the level of ferritin protein could control the pool of intracellular iron. The present results indicate that iron homeostasis in lens epithelial cells is affected mainly by changes in apoferritin synthesis, which is greatly stimulated by ascorbic acid, rather than by altering the rate of protein degradation, which is very slow in these cells under all circumstances. Ferric ammonium citrate activates iron uptake from transferrin in a wide range of cell lines by generation of free radicals. Ferric ammonium citrate also increased iron uptake from Tf in lens epithelial cells. Ferric ammonium citrate treated cells incorporated 5 times more iron and deposited 2 times more iron into ferritin than control cells. Increased incorporation

  17. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals

    PubMed Central

    1995-01-01

    Cataract is a major ocular disease that causes blindness in many developing countries of the world. It is well established that various factors such as oxidative stress, UV, and other toxic agents can induce both in vivo and in vitro cataract formation. However, a common cellular basis for this induction has not been previously recognized. The present study of lens epithelial cell viability suggests such a general mechanism. When lens epithelial cells from a group of 20 cataract patients 12 to 94 years old were analyzed by terminal deoxynucleotidyl transferase (TdT) labeling and DNA fragmentation assays, it was found that all of these patients had apoptotic epithelial cells ranging from 4.4 to 41.8%. By contrast, in eight normal human lenses of comparable age, very few apoptotic epithelial cells were observed. We suggest that cataract patients may have deficient defense systems against factors such as oxidative stress and UV at the onset of the disease. Such stress can trigger lens epithelial cell apoptosis that then may initiate cataract development. To test this hypothesis, it is also demonstrated here that hydrogen peroxide at concentrations previously found in some cataract patients induces both lens epithelial cell apoptosis and cortical opacity. Moreover, the temporal and spatial distribution of induced apoptotic lens epithelial cells precedes development of lens opacification. These results suggest that lens epithelial cell apoptosis may be a common cellular basis for initiation of noncongenital cataract formation. PMID:7790371

  18. Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

    PubMed Central

    Fujimichi, Yuki; Hamada, Nobuyuki

    2014-01-01

    Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that

  19. Elevated tropomyosin expression is associated with epithelial–mesenchymal transition of lens epithelial cells

    PubMed Central

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelial–mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis. PMID:23205574

  20. Development of an accommodating intra-ocular lens--in vitro prevention of re-growth of pig and rabbit lens capsule epithelial cells.

    PubMed

    van Kooten, Theo G; Koopmans, Steven; Terwee, Thom; Norrby, Sverker; Hooymans, J M M; Busscher, Henk J

    2006-11-01

    Cataract surgery is routinely performed to replace the clouded lens by a rigid polymeric intra-ocular lens unable to accommodate. By implanting a silicone gel into an intact capsular bag the accommodating properties of the natural lens can be maintained or enhanced. The implantation success of accommodating lenses is hampered by the occurrence of capsular opacification (PCO) due to lens epithelial cell (LEC) growth. In order to prevent LEC proliferation, a treatment regime using actinomycin D, cycloheximide and water was developed. The effectiveness of treatment was analyzed using an in vitro, MTT-based cell culture system and an ex vivo pig eye model in which the implanted lens-in-the-bag is cultured as a whole. LEC were exposed to treatment solutions for 5 min, then the cells were allowed to recover and to re-colonize the substratum. MTT conversion by cells was transiently inhibited by cycloheximide dissolved in water and by water alone. Exposure to actinomycin D resulted in a lasting inhibition of MTT conversion and consequently cell proliferation. These in vitro data could not be fully reproduced in the ex vivo pig eye model due to essential differences between both models. Treatment with actinomycin D containing solutions, however, resulted in a nearly complete absence of cells on the capsular wall. The pig eye model is a promising approach to further evaluate the effects of peri-surgical treatment during the accommodating intra-ocular lens implantation.

  1. Comparative photodynamic effect of rose bengal, Erythrosin B, and dihematoporphyrin ethers on lens epithelial cells

    NASA Astrophysics Data System (ADS)

    Takesue, Yoshiko; Mui, Mei M.; Hachiya, Takahiko; Parel, Jean-Marie A.

    1993-06-01

    The proliferation of residual lens epithelial cells (LEC) is responsible for reopacification of the lens capsule and loss of visual acuity after cataract surgery. Photodynamic therapy (PDT) using Di-Hematoporphyrin Ether (DHE) was shown to kill cultured LEC. We studied 2 synthetic photodynamic drugs: Rose Bengal (RB) and Erythrosin B (EB) to determine the minimal PDT parameters. LEC located on the anterior capsule of freshly explanted rabbit lenses were subcultured for the following studies: (1) Dye uptake was evaluated by cellular fluorescence. The minimum concentrations for RB and EB after 1 minute of exposure were 1 (mu) g/ml, and 50 (mu) g/ml; (2) Cytotoxicity was evaluated by reculturing the cells, and was detectable with concentrations of 200 (mu) g/ml RB or 2000 (mu) g/ml EB; (3) The PDT effect was evaluated using concentrations of 1 to 25 (mu) g/ml of RB and 1 to 2000 (mu) g/ml of EB. The minimum parameters for PDT of cultured LEC were 0.5 (mu) g/ml of RB and 10 (mu) g/ml of EB with a 60 second 0.5 W/cm2 irradiation using a 514.5 nm Argon laser. To mimic the in vivo setting, LEC attached to the anterior lens capsule were incubated with EB to evaluate dye uptake. The minimum parameters were 100 (mu) g/ml and 7 minutes of exposure. RB was found most effective and may be useful, but EB was found safer and has potential for conjugation with LEC antibodies for the prevention of secondary cataracts.

  2. Protocatechualdehyde prevents methylglyoxal-induced mitochondrial dysfunction and AGEs-RAGE axis activation in human lens epithelial cells.

    PubMed

    Wang, Yue-Hua; Han, Yu-Pei; Yu, Hai-Tao; Pu, Xiao-Ping; Du, Guan-Hua

    2014-09-05

    Methylglyoxal (MGO), a glucose derived dicarbonyl intermediate, is a major precursor of advanced glycation end products (AGEs) which have been linked to the development of diabetic cataract. Protocatechualdehyde (PCA), a phenolic acid compound, is found in the roots of Salvia miltiorrhiza. This study was to investigate the effect of PCA against MGO-induced cytotoxicity in human lens epithelial cells (SRA01/04 cells) and the possible involved molecular mechanism. The results showed that PCA alleviated MGO-induced mitochondrial dysfunction and apoptosis in SRA01/04 cells. Furthermore, PCA was capable of inhibiting MGO-mediated AGEs formation and blocking receptor of AGEs expression in SRA01/04 cells. It is concluded that PCA could be useful in attenuation of MGO-induced cell damage and the possible mechanism is involved in modulating AGEs-receptor of AGEs axis in human lens epithelial cells, which suggests that PCA has a potential protective effect on diabetic cataract.

  3. Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions

    PubMed Central

    Tanti, N.C.; Jones, L.; Sheardown, H.

    2010-01-01

    Purpose Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate  (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed

  4. Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions.

    PubMed

    Gorbet, M B; Tanti, N C; Jones, L; Sheardown, H

    2010-02-19

    Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of beta(1) and alpha(3) integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These

  5. Micropatterned Protective Membranes Inhibit Lens Epithelial Cell Migration in Posterior Capsule Opacification Model.

    PubMed

    Magin, Chelsea M; May, Rhea M; Drinker, Michael C; Cuevas, Kevin H; Brennan, Anthony B; Reddy, Shravanthi T

    2015-03-01

    To evaluate the ability of Sharklet (SK) micropatterns to inhibit lens epithelial cell (LEC) migration. Sharklet Technologies, Inc. (STI) and InSight Innovations, LLC have proposed to develop a Sharklet-patterned protective membrane (PM) to be implanted in combination with a posterior chamber intraocular lens (IOL) to inhibit cellular migration across the posterior capsule, and thereby reduce rates of posterior capsular opacification (PCO). A variety of STI micropatterns were evaluated versus smooth (SM) controls in a modified scratch wound assay for the ability to reduce or inhibit LEC migration. The best performing topography was selected, translated to a radial design, and applied to PM prototypes. The PM prototypes were tested in an in vitro PCO model for reduction of cell migration behind an IOL versus unpatterned prototypes and IOLs with no PM. In both assays, cell migration was analyzed with fluorescent microscopy. All SK micropatterns significantly reduced LEC migration compared with SM controls. Micropatterns that protruded from the surface reduced migration more than recessed features. The best performing micropattern reduced LEC coverage by 80%, P = 0.0001 (ANOVA, Tukey Test). Micropatterned PMs reduced LEC migration in a PCO model by 50%, P = 0.0005 (ANOVA, Tukey Test) compared with both IOLs with no PM and IOLs with SM PMs. Collectively, in vitro results indicate the implantation of micropatterned PMs in combination with posterior chamber IOLs could significantly reduce rates of clinically relevant PCO. This innovative technology is a globally accessible solution to high PCO rates. A novel IOL incorporating the SK micropattern in a membrane design surrounding the optic may help increase the success of cataract surgery by reducing secondary cataract, or PCO.

  6. Micropatterned Protective Membranes Inhibit Lens Epithelial Cell Migration in Posterior Capsule Opacification Model

    PubMed Central

    Magin, Chelsea M.; May, Rhea M.; Drinker, Michael C.; Cuevas, Kevin H.; Brennan, Anthony B.; Reddy, Shravanthi T.

    2015-01-01

    Purpose To evaluate the ability of Sharklet (SK) micropatterns to inhibit lens epithelial cell (LEC) migration. Sharklet Technologies, Inc. (STI) and InSight Innovations, LLC have proposed to develop a Sharklet-patterned protective membrane (PM) to be implanted in combination with a posterior chamber intraocular lens (IOL) to inhibit cellular migration across the posterior capsule, and thereby reduce rates of posterior capsular opacification (PCO). Methods A variety of STI micropatterns were evaluated versus smooth (SM) controls in a modified scratch wound assay for the ability to reduce or inhibit LEC migration. The best performing topography was selected, translated to a radial design, and applied to PM prototypes. The PM prototypes were tested in an in vitro PCO model for reduction of cell migration behind an IOL versus unpatterned prototypes and IOLs with no PM. In both assays, cell migration was analyzed with fluorescent microscopy. Results All SK micropatterns significantly reduced LEC migration compared with SM controls. Micropatterns that protruded from the surface reduced migration more than recessed features. The best performing micropattern reduced LEC coverage by 80%, P = 0.0001 (ANOVA, Tukey Test). Micropatterned PMs reduced LEC migration in a PCO model by 50%, P = 0.0005 (ANOVA, Tukey Test) compared with both IOLs with no PM and IOLs with SM PMs. Conclusions Collectively, in vitro results indicate the implantation of micropatterned PMs in combination with posterior chamber IOLs could significantly reduce rates of clinically relevant PCO. This innovative technology is a globally accessible solution to high PCO rates. Translational Relevance A novel IOL incorporating the SK micropattern in a membrane design surrounding the optic may help increase the success of cataract surgery by reducing secondary cataract, or PCO. PMID:25883876

  7. Serum and aqueous xanthine oxidase levels, and mRNA expression in anterior lens epithelial cells in pseudoexfoliation.

    PubMed

    Simavli, Huseyin; Tosun, Mehmet; Bucak, Yasin Y; Erdurmus, Mesut; Ocak, Zeynep; Onder, Halil I; Acar, Muradiye

    2015-07-01

    The aim of this study was to determine serum and aqueous xanthine oxidase (XO) levels, and mRNA expression in anterior lens epithelial cells in pseudoexfoliation (PEX). In this prospective study, serum, aqueous and anterior lens capsules were taken from 21 patients with PEX and 23 normal subjects who had undergone routine cataract surgery. Serum and aqueous XO levels were analyzed using the colorimetric method. mRNA expression of XO in anterior lens epithelial cells was evaluated using reverse transcription polymerase chain reaction analysis. Serum XO levels (means ± standard deviations) were 207.0 ± 86.1 IU/mL and 240.6 ± 114.1 IU/mL in the normal and PEX groups, respectively (p = 0.310). Aqueous XO levels (means ± standard deviations) were 65.5 ± 54.3 IU/mL in the normal group and 130.5 ± 117.4 IU/mL in the PEX group (p = 0.028). There was a 2.9 fold decrease in mRNA expression in anterior lens epithelial cells of PEX, which is significantly lower than the normal group (p = 0.01). Higher aqueous XO levels lacking associated different serum XO suggests higher oxidative stress in the aqueous. Higher aqueous XO levels in PEX with decreased mRNA expression in anterior lens epithelial cells indicate possible overexpression of XO in other structures related to the aqueous.

  8. Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells

    PubMed Central

    Tanti, N.C.; Crockett, B.; Mansour, L.; Jones, L.

    2011-01-01

    Purpose Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. Methods The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. Results A significant reduction in viability (between 30 to 50%) was observed with cells exposed to lenses soaked in ReNu and OFX. A significant downregulation of α3 and β1 integrins, with integrin expression ranging from 60% to 75% of control (cells with no lens), was also observed with OFX and ReNu-soaked lenses. With the exception of BA, all other lenses soaked in OFX resulted in significant caspase activation, whereby over 18% of cells stained positive for caspases. Minimal caspase activation was observed in cells exposed to ReNu and Solo soaked lenses. For both OFX and ReNu, exposing cells to at least a 5% dilution had a significant effect on viability and integrin expression. While Complete and Solo did not lead to reduction in viability, cells exposed to a 10% dilution showed reduced integrin expression down to less than 70% of control value. Comparing cell response to diluted MPS solutions and various MPS-soaked lenses showed that it is not possible to reliably use cell response to MPS dilution alone to assess MPS

  9. Alpha lipoic acid changes iron uptake and storage in lens epithelial cells.

    PubMed

    Goralska, Malgorzata; Dackor, Ryan; Holley, Benjamin; McGahan, M Christine

    2003-02-01

    Alpha lipoic acid (LA) is a cofactor in mitochondrial dehydrogenase complexes. Previous studies have shown that when administered exogenously LA has antioxidant properties, which include free radical scavenging, metal chelation and regeneration of other antioxidants. The cells convert LA into dihydroplipoic acid (DHLA), which in the presence of iron can act as a prooxidant. In vitro DHLA reduces Fe(+3) to Fe(+2) and removes iron from ferritin, increasing the risk of Fe catalyzed free radical formation. In the present study we examined the in vivo effects of lipoic acid treatment on Fe metabolism in cultured lens epithelial cells, and found that LA decreases Fe uptake from transferrin, increases Fe deposition into ferritin and increases the concentration of this protein. When administered together with ascorbic acid, lipoic acid changes the characteristic heavy to light chain ratio of ferritin makeup. The decreased Fe uptake and increased storage diminishes the size of the cytosolic highly reactive Fe pool (LIP). These changes are associated with increased cell resistance to H(2)O(2) challenge. Therefore, LA may reduce the risk of Fe induced oxidative damage and also might be useful as a treatment of Fe overload. Copyright 2003 Elsevier Science Ltd.

  10. Effect of ultraviolet A exposure on transport of compatible organic osmolytes in human lens epithelial cells.

    PubMed

    Wu, D Y; Zhang, J S

    2015-05-18

    Compatible organic osmolytes, such as betaine, myoinositol, and taurine, are involved in antioxidant defense, protein stabilization, and stress responses. This osmolyte strategy requires the expression of specific osmolyte transporters such as betaine (BGT-1), myoinositol (SMIT), and taurine (TAUT). In contrast to the kidney, keratinocytes, and neural cells, few studies have examined osmolytes in human lens epithelial cells (HLECs). We examined the expression of mRNA specific for BGT-1, SMIT, and TAUT in HLECs. In comparison to normoosmotic (305 mOsM) controls, there was a 3-5-fold time-dependent reaction of BGT-1, SMIT, and TAUT mRNA levels in HLECs exposed to hyperosmotic stress (405 mOsM). Maximal responses were obtained for BGT-1, SMIT, and TAUT mRNA expression after 3, 24 and 9 h of hyperosmotic exposure, respectively. This expression was correlated with increased osmolyte uptake. In contrast, hypoosmotic (205 mOsM) stimulation led to a significant efflux of osmolytes. Exposure to ultraviolet A (340-400 nm) radiation significantly stimulated osmolyte uptake. Increased osmolyte uptake was associated with upregulation of mRNA steady-state levels for osmolyte transporters in irradiated cells. These results demonstrate that ultraviolet A radiation leads to the accumulation of compatible organic osmolytes in HLECs as hyperosmotic pressure, which can maintain cellular environmental homeostasis.

  11. Proton Irradiation Alters Expression of FGF-2 In Human Lens Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.

    1999-01-01

    We are investigating a role for proton radiation-induced changes in FGF-2 gene expression as part of the mechanism(s) underlying lens cell injury. Radiation injury to the human lens is associated with the induction of cataract following exposure to protons.

  12. RhoA/ROCK Signaling Regulates TGFβ-Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells through MRTF-A

    PubMed Central

    Korol, Anna; Taiyab, Aftab; West-Mays, Judith A

    2016-01-01

    Transforming growth factor (TGF)-β–induced epithelial-mesenchymal transition (EMT) leads to the formation of ocular fibrotic pathologies, such as anterior subcapsular cataract and posterior capsule opacification. Remodeling of the actin cytoskeleton, mediated by the Rho family of GTPases, plays a key role in EMT. However, how actin dynamics affect downstream markers of EMT has not been fully determined. Our previous work suggests that myocardin-related transcription factor A (MRTF-A), an actin-binding protein, might be an important mediator of TGFβ-induced EMT in lens epithelial cells. The aim of the current study was to determine the requirement of RhoA/ROCK signaling in mediating TGFβ-induced nuclear accumulation of MRTF-A and ultimate expression of α-smooth muscle actin (αSMA), a marker of a contractile myofibroblast phenotype. Using rat lens epithelial explants, we demonstrate that ROCK inhibition using Y-27632 prevents TGFβ-induced nuclear accumulation of MRTF-A, E-cadherin/β-catenin complex disassembly, and αSMA expression. Using a novel inhibitor specifically targeting MRTF-A signaling, CCG-203971, we further demonstrate the requirement of MRTF-A nuclear localization and activity in the induction of αSMA expression. Overall, our findings suggest that TGFβ-induced cytoskeletal reorganization through RhoA/ROCK/MRTF-A signaling is critical to EMT of lens epithelial cells. PMID:27704140

  13. Effect of superposed electromagnetic noise on DNA damage of lens epithelial cells induced by microwave radiation.

    PubMed

    Yao, Ke; Wu, Wei; Yu, Yibo; Zeng, Qunli; He, Jiliang; Lu, Deqiang; Wang, Kaijun

    2008-05-01

    To investigate the influence of the 1.8-GHz radiofrequency fields (RFs) of the Global System for Mobile Communications on DNA damage, intracellular reactive oxygen species (ROS) formation, cell cycle, and apoptosis in human lens epithelial cells (hLECs) and whether the effects induced by RF could be blocked by superposing of electromagnetic noise. After 24-hour intermittent exposure at the specific absorption rate of 1 W/kg, 2 W/kg, 3 W/kg, and 4 W/kg, the DNA damage of hLECs was examined by alkaline comet assay and immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (gammaH2AX) foci, respectively. ROS production was quantified by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell cycle and cell apoptosis were determined by flow cytometry. DNA damage examined by alkaline comet assay was significantly increased after 3 W/kg and 4 W/kg radiation (P < 0.05), whereas the double-strand breaks (DSBs) evaluated by gammaH2AX foci were significantly increased only after 4 W/kg radiation (P < 0.05). Significantly elevated intracellular ROS levels were also detected in the 3-W/kg and 4-W/kg groups (P < 0.05). After exposure to 4 W/kg for 24 hours, hLECs exhibited significant G(0)/G(1) arrest (P < 0.05). There was no detectable difference in cell apoptosis between the microwave radiation and sham exposure groups (P > 0.05). All the effects mentioned were blocked when the RF was superposed with 2 muT electromagnetic noise. Microwave radiation induced hLEC DNA damage after G(0)/G(1) arrest does not lead to cell apoptosis. The increased ROS observed may be associated with DNA damage. Superposed electromagnetic noise blocks microwave radiation-induced DNA damage, ROS formation, and cell cycle arrest.

  14. In vitro investigation of ultrasound-induced oxidative stress on human lens epithelial cells.

    PubMed

    Rwei, Patrick; Alex Gong, Cihun-Siyong; Luo, Li-Jyuan; Lin, Meng-Bo; Lai, Jui-Yang; Liu, Hao-Li

    2017-01-22

    The effect of ultrasound exposure on human lens epithelial cells (HLE-B3) was investigated in vitro, specifically on the generation of oxidative stress upon ultrasound application using various clinically-relevant settings. In addition to ultrasound-induced heat effects, oxidative stress has been recently proposed as one of the main mechanisms for ultrasound-induced effects on human cells. In this work, the levels of biocompatibility and generation of oxidative stress by exposure of ultrasound to HLE-B3 were evaluated quantitatively and qualitatively by the MTT assay, Live/Dead assay, reactive oxygen species (ROS) and intracellular calcium level. Oxidative stress induction is traditionally achieved through administrations of H2O2 and thus the administration of H2O2 was used as the positive control group for comparison herein. Concerning the administrations of H2O2 are considered invasive and may potentially have side effects, ultrasound as physical stimulation could be a safer and non-invasive method to induce similar oxidative stress environments. The effect of ultrasound on cell viability and induction of oxidative stress increases with ultrasound intensity. The result reveals that the continuous ultrasound has a positive impact on the oxidative stress levels but does negatively on the cell viability, as compared to the pulsed ultrasound. Furthermore, our work demonstrates that the exposure of 58 kPa continuous ultrasound without microbubbles can maintain acceptable cell viability and produce oxidative stress effects similar to the traditional administrations of H2O2. In summary, exposure of ultrasound can generate oxidative stress comparable to traditional administrations of H2O2. The effect of generating oxidative stress is adjustable through ultrasound parameters, including the pulsed or continuous wave, the intensity of ultrasound and addition of microbubbles.

  15. Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.

    PubMed

    Lauf, Peter K; Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C

    2008-03-01

    This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.

  16. Distribution, Molecular Structure and Functional Analysis of Carnitine Transporter (SLC22A5) in Canine Lens Epithelial Cells

    PubMed Central

    OCHIAI, Hideharu; KANEMAKI, Nobuyuki; SATO, Reiichiro; ONDA, Ken

    2014-01-01

    While carnitine has been reported to have an anti-oxidative role on the ocular surface, there has been no report on the existence of a carnitine transporter (SLC22A5) in the lens. Therefore, we investigated the carnitine transport activity of canine lens epithelial cells (LEC) and determined the molecular structure of canine SLC22A5. The carnitine transport activity was 7.16 ± 0.48 pmol/mg protein/30 min. Butyrobetaine, the analogue of carnitine, reduced 30% of the activity at 50 µM. A coding sequence of canine carnitine transporter was 1694 bp long and was predicted to encode 557 amino acid polypeptides. The deduced amino acid sequence of canine carnitine transporter showed >80% similarity to that of mouse and human. Western blot analysis detected the band at 60 kDa in the membrane of lens epithelial cells. The high content of carnitine in the lens is possibly transported from aqueous humor by SLC22A5. PMID:25048262

  17. Thioredoxin Binding Protein-2 Regulates Autophagy of Human Lens Epithelial Cells under Oxidative Stress via Inhibition of Akt Phosphorylation

    PubMed Central

    Yao, Ke; Zhang, Yidong; Chen, Guangdi; Lai, Kairan; Yin, Houfa

    2016-01-01

    Oxidative stress plays an essential role in the development of age-related cataract. Thioredoxin binding protein-2 (TBP-2) is a negative regulator of thioredoxin (Trx), which deteriorates cellular antioxidant system. Our study focused on the autophagy-regulating effect of TBP-2 under oxidative stress in human lens epithelial cells (LECs). Human lens epithelial cells were used for cell culture and treatment. Lentiviral-based transfection system was used for overexpression of TBP-2. Cytotoxicity assay, western blot analysis, GFP/mCherry-fused LC3 plasmid, immunofluorescence, and transmission electronic microscopy were performed. The results showed that autophagic response of LECs with increased LC3-II, p62, and GFP/mCherry-LC3 puncta (P < 0.01) was induced by oxidative stress. Overexpression of TBP-2 further strengthens this response and worsens the cell viability (P < 0.01). Knockdown of TBP-2 attenuates the autophagic response and cell viability loss induced by oxidative stress. TBP-2 mainly regulates autophagy in the initiation stage, which is mTOR-independent and probably caused by the dephosphorylation of Akt under oxidative stress. These findings suggest a novel role of TBP-2 in human LECs under oxidative stress. Oxidative stress can cause cell injury and autophagy in LECs, and TBP-2 regulates this response. Hence, this study provides evidence regarding the role of TBP-2 in lens and the possible mechanism of cataract development. PMID:27656263

  18. Sprouty2 Suppresses Epithelial-Mesenchymal Transition of Human Lens Epithelial Cells through Blockade of Smad2 and ERK1/2 Pathways

    PubMed Central

    Chen, Chuan; Chen, Xiaoyun; Qin, Yingyan; Qu, Bo; Luo, Lixia; Lin, Haotian; Wu, Mingxing; Chen, Weirong; Liu, Yizhi

    2016-01-01

    Transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a key role in the pathogenesis of anterior subcapsular cataract (ASC) and capsule opacification. In mouse lens, Sprouty2 (Spry2) has a negative regulatory role on TGFβ signaling. However, the regulation of Spry2 during ASC development and how Spry2 modulates TGFβ signaling pathway in human LECs have not been characterized. Here, we demonstrate that Spry2 expression level is decreased in anterior capsule LECs of ASC patients. Spry2 negatively regulates TGFβ2-induced EMT and migration of LECs through inhibition of Smad2 and ERK1/2 phosphorylation. Also, blockade of Smad2 or ERK1/2 activation suppresses EMT caused by Spry2 downregulation. Collectively, our results for the first time show in human LECs that Spry2 has an inhibitory role in TGFβ signaling pathway. Our findings in human lens tissue and epithelial cells suggest that Spry2 may become a novel therapeutic target for the prevention and treatment of ASC and capsule opacification. PMID:27415760

  19. Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells - Relevance to Capsular Opacification.

    PubMed

    Recek, Nina; Andjelić, Sofija; Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

    2016-01-01

    Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation.

  20. Differential degradation of ferritin H- and L-chains: accumulation of L-chain-rich ferritin in lens epithelial cells.

    PubMed

    Goralska, Malgorzata; Nagar, Steven; Fleisher, Lloyd N; McGahan, M Christine

    2005-10-01

    The storage of iron by ferritin is determined by tissue-specific composition of its 24 subunits, which are designated as either heavy (H) or light (L). For a better understanding of how lens epithelial cells regulate their ferritin subunit makeup, the degradation pattern of each subunit type was analyzed. In addition, age-related changes in ferritin concentration and subunit makeup were determined. Ferritin turnover in primary cultures of canine lens epithelial cells was determined by metabolic labeling with [(35)S]-methionine. Transient transfection with vectors containing coding sequences for either H- or L-chains were used to modify ferritin subunit makeup. Ferritin concentration was measured by ELISA. Immunodetection and fluorescence immunocytochemistry were used to study age-related changes in ferritin chain concentration. Inhibition of the proteasomal protein degradation pathway by clastolactacystin-beta-lactone had no effect on ferritin degradation, whereas inhibition of lysosomal degradation markedly increased ferritin levels, confirming that this system is involved in ferritin turnover. H-chain ferritin degraded at a faster rate than the L-chain. L-chain-rich ferritin in L-chain-transfected cells formed inclusion bodies that were localized to the cytosol. Similar inclusion bodies were found in older lens cells kept in cell culture for more than 8 days. Steady degradation of H-chain ferritin contributed to the maintenance of a constant level of this chain within the lens epithelial cells. In contrast, slower turnover of the L-chain resulted in accumulation of L-chain-enriched ferritin associated with cytoplasmic inclusion bodies. These L-chain-containing inclusion bodies were found in the cytosol of cells overexpressing L-ferritin chain and in nontransfected cells maintained in culture for 8 to 35 days. Overexpression of the L-chain has been associated with the formation of premature cataracts in humans with hereditary hyperferritinemia cataract syndrome

  1. Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth

    PubMed Central

    Liu, Jin-Lu; Zhang, Wen-Ji; Li, Xue-Dong; Yang, Na; Pan, Wei-San; Kong, Jun; Zhang, Jin-Song

    2016-01-01

    AIM To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein (Gen-NLC) to inhibit human lens epithelial cells (HLECs) proliferation. METHODS Gen-NLC was made by melt emulsification method. The morphology, particle size (PS), zeta potentials (ZP), encapsulation efficiency (EE) and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier (NLC), genistein (Gen) and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8 (CCK-8) assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence analyses. RESULTS The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was -7.14±0.38 mV and the EE of Gen in the nanoparticles was 92.3%±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The mRNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group. CONCLUSION Sustained drug release by Gen-NLCs may impede HLEC growth. PMID:27275415

  2. Molecular cloning of canine excitatory amino acid transporter 5 and its detection in primary lens epithelial cells.

    PubMed

    Ochiai, Hideharu; Saito, Miyoko; Maruo, Takuya; Kanemaki, Nobuyuki

    2010-01-01

    The full-length cDNA sequence of canine excitatory amino acid transporter (EAAT) 5 was determined in samples taken from the canine retina. The sequence was 2,467 bp long and was predicted to encode the 560 amino acid polypeptides. The deduced amino acid sequence of canine EAAT5 showed similarities of 91.8 and 92.7% to those of humans and rats, respectively. In canine, EAAT5 has a 49.4% identity with EAAT1, 43.7% with EAAT2, 46.4% with EAAT3, and 45.7% with EAAT4. RT-PCR analysis revealed EAAT5 expression in primary lens epithelial cell culture and the cerebellum, and Western blot analysis detected a single band of 60 kDa which confirmed EAAT5 protein expression in these cells. In addition, all subtypes of EAAT were detected in canine lens epithelial cells, indicating the pivotal role of EAATs in supplying glutamate, the precursor of antioxidant glutathione in the lens.

  3. Cytotoxicity testing of multipurpose contact lens solutions using monolayer and stratified cultures of human corneal epithelial cells.

    PubMed

    Lim, Mary J; Hurst, Rachel K; Konynenbelt, B J; Ubels, John L

    2009-11-01

    To investigate in vitro cytotoxicity of five marketed multipurpose contact lens solutions (MPS) on the morphology, viability, and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Cells were exposed to MPS for 10, 20, or 60 minutes. In monolayer cultures, effects of the MPS on cell morphology were observed using Hoffman modulation contrast microscopy. Cell proliferation after exposure to MPS was evaluated and cytotoxicity of the MPS was determined using a live/dead cell assay and flow cytometry. In stratified cultures, multilayer corneal epithelial constructs were established on membrane inserts. Effects of the MPS on the morphology and barrier function of stratified cultures were determined using microscopy, a fluorescein permeability test, and measurement of trans-epithelial resistance. In monolayer cultures, none of the MPS damaged cells during a 10-min exposure. All of the MPS had varying time-dependent adverse effects on cell morphology, viability, and proliferation during 20- and 60-min exposures. In stratified cultures, none of the MPS had an adverse effect on the structure or barrier function of stratified cultures. Monolayer cultures are highly sensitive to damage by MPS. In contrast, because stratified human corneal-limbal epithelial cultures are resistant to adverse effects of MPS, it is suggested that models that simulate the stratified structure of the corneal epithelium should be used for in vitro toxicologic testing. Caution should be used when interpreting such studies, because in vitro tests may not be predictive of clinical responses to contact lens products that are known to be safe when used as directed.

  4. Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.

    PubMed

    De María, Alicia; Arruti, Cristina

    2003-12-19

    Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation.

  5. Phycocyanin may suppress D-galactose-induced human lens epithelial cell apoptosis through mitochondrial and unfolded protein response pathways.

    PubMed

    Ou, Yu; Yuan, Zhijun; Li, Kepeng; Yang, Xuegan

    2012-11-23

    Apoptosis of lens epithelial cell (LEC) plays an important role in cataract formation, and its prevention may be one of the therapeutic strategies in treating cataract. This study used human lens epithelial cell (hLEC) line SRA01/04 to investigate the protective effect and mechanism of phycocyanin on glactose-induced apoptosis in hLEC. hLECs were cultured in D/F(12)-10% FBS medium containing 125mM d-galactose with or without phycocyanin. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was elevated with Wright-Giemsa staining, AO/EB double staining, and DNA fragmentation assay. Mitochondrial apoptosis-associated molecules and unfolded protein response-associated molecules from cultured SRA01/04 cells were quantified using protein blot analysis. The results demonstrated that phycocyanin suppressed SRA01/04 cells' morphologic changes and apoptosis induced by d-galactose, inhibited the expression and activation of caspase 3, alternated the Bax/Bcl-2 ratio, and down-regulated the level of p53, GRP78, and CHOP in d-galactose-treated SRA01/04 cells. These results suggest that phycocyanin might suppress d-galactose-induced hLEC apoptosis through two pathways: mitochondrial pathway, involving p53 and Bcl-2 family protein expression, and unfolded protein response pathway, involving GRP78 and CHOP expression.

  6. Lens epithelium-derived growth factor relieves transforming growth factor-beta1-induced transcription repression of heat shock proteins in human lens epithelial cells.

    PubMed

    Sharma, Preeti; Fatma, Nigar; Kubo, Eri; Shinohara, Toshimichi; Chylack, Leo T; Singh, Dhirendra P

    2003-05-30

    Lens epithelium-cell derived growth factor (LEDGF) is a transcriptional activator. It protects the cells by binding to cis-stress response ((A/T)GGGG(T/A)), and heat shock (HSE; nGAAn) elements in the stress genes and activating their transcription. Transforming growth factor-beta (TGF-beta) has been implicated in the control of tissue homeostasis, terminal differentiation, and apoptosis. Here we provide evidence that TGF-beta1 down-regulates LEDGF expression and diminishes its affinity for DNA during TGF-beta1-induced phenotypic changes and apoptosis in human lens epithelial cells. Surprisingly, TGF-beta1 treatment for 48 h markedly decreased the LEDGF, Hsp27, and alphaB-crystallin promoter activities with the decrease of abundance of LEDGF mRNA and protein. Deletion mutants of the LEDGF promoter showed that one TGF-beta1 inhibitory element (TIE) like sequence nnnTTGGnnn (-444 to -433) contributed to this negative regulation. Mutation of TIE (TTGG to TATT) abolished the down-regulation of the LEDGF promoter. Gel mobility and supershift assays showed that LEDGF in the nuclear extracts of TGF-beta1-treated human lens epithelial cells did not bind to stress-response elements and HSE. The TGF-beta1-induced down-regulation of LEDGF, Hsp27, and alphaB-crystallin promoters activity was reversed by cotransfection with a plasmid expressing LEDGF. Because overexpression of LEDGF was able to relieve TGF-beta1 and/or stress-induced changes, it would be a candidate molecule to postpone age-related degenerating disorders.

  7. The role of metallothionein IIa in defending lens epithelial cells against cadmium and TBHP induced oxidative stress

    PubMed Central

    Hawse, John R.; Padgaonkar, Vanita A.; Leverenz, Victor R.; Pelliccia, Sara E.; Kantorow, Marc; Giblin, Frank J.

    2007-01-01

    Purpose Heavy metals and other forms of oxidative stress have been implicated as key factors in the formation of age-related cataract in humans. Metallothioneins are a group of proteins known to play important roles in defending cells against the cytotoxic effects of heavy metals. However, little is known about their involvement in defending against other forms of oxidative stress. Here, we examined the ability of metallothionein IIa (MTIIa) to protect human lens epithelial cells against cadmium and tertiary butyl hydroperoxide (TBHP)-induced oxidative stress. Methods MTIIa over-expressing human lens epithelial cells (SRA01/04) were created by retroviral mediated gene transfer. Normal and MTIIa over-expressing cells were exposed to various concentrations of cadmium and TBHP and subsequently monitored for cell death, changes in cell phenotype and differences in growth rate. In addition, expression levels of three other important antioxidant genes, heme oxygenase-1, thioredoxin reductase-1, and manganese superoxide dismutase were monitored by real-time RT-PCR following exposure to TBHP. Results Analysis of the over expressing cell lines revealed an approximate 3–4 fold increase in MTIIa expression relative to control cells, resulting in as much as 20% protection against cadmium-induced oxidative stress (p<0.001). The MTIIa over expressing cells were also significantly more resistant to TBHP treatment while control cells exhibited significant shrinking and rounding-up following 3–6 h TBHP treatment, no changes were observed in TBHP-treated over expressing cells. When control cells were treated for 3 h or overnight with TBHP, 40–45% cell death occurred by day three. However, no cell death was observed at this time for the treated MTIIa over-expressing cell line. In addition, TBHP induced the expression of MTIIa, heme oxygenase-1, thioredoxin reductase-1, and MnSOD in both normal and MTIIa over-expressed cell lines. Interestingly the latter three genes were

  8. Adapting biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels for pigment epithelial cell encapsulation and lens regeneration.

    PubMed

    Zhang, Mimi W; Park, Hansoo; Guo, Xuan; Nakamura, Kenta; Raphael, Robert M; Kasper, F Kurtis; Mikos, Antonios G; Tsonis, Panagiotis A

    2010-04-01

    This study investigated the encapsulation of newt iris pigment epithelial cells (PECs), which have the ability to regenerate a lens by trans-differentiation in vivo, within a biodegradable hydrogel of oligo(poly(ethylene glycol) fumarate) crosslinked with poly(ethylene glycol)-diacrylate. Hydrogel beads of initial diameter of 1 mm were fabricated by a molding technique. The swelling ratio and degradation rate of the hydrogel beads decreased with increasing crosslinking ratios. Confocal microscopy confirmed the cytocompatibility of crosslinking hydrogel formulations as evidenced by the viability of an encapsulated model cell line within a crosslinked hydrogel bead. Hydrogel beads encapsulating iris PECs were also implanted into lentectomized newts in vivo; histological evaluation of explants after 30 days revealed a regenerated lens, thus demonstrating that the presence of degrading hydrogel did not adversely affect lens regeneration. The results of this study suggest the potential of a method for lens regeneration involving oligo(poly(ethylene glycol) fumarate) hydrogels for iris PEC encapsulation and transplantation.

  9. Adapting Biodegradable Oligo(Poly(Ethylene Glycol) Fumarate) Hydrogels for Pigment Epithelial Cell Encapsulation and Lens Regeneration

    PubMed Central

    Zhang, Mimi W.; Park, Hansoo; Guo, Xuan; Nakamura, Kenta; Raphael, Robert M.; Kasper, F. Kurtis

    2010-01-01

    This study investigated the encapsulation of newt iris pigment epithelial cells (PECs), which have the ability to regenerate a lens by trans-differentiation in vivo, within a biodegradable hydrogel of oligo(poly(ethylene glycol) fumarate) crosslinked with poly(ethylene glycol)-diacrylate. Hydrogel beads of initial diameter of 1 mm were fabricated by a molding technique. The swelling ratio and degradation rate of the hydrogel beads decreased with increasing crosslinking ratios. Confocal microscopy confirmed the cytocompatibility of crosslinking hydrogel formulations as evidenced by the viability of an encapsulated model cell line within a crosslinked hydrogel bead. Hydrogel beads encapsulating iris PECs were also implanted into lentectomized newts in vivo; histological evaluation of explants after 30 days revealed a regenerated lens, thus demonstrating that the presence of degrading hydrogel did not adversely affect lens regeneration. The results of this study suggest the potential of a method for lens regeneration involving oligo(poly(ethylene glycol) fumarate) hydrogels for iris PEC encapsulation and transplantation. PMID:19514850

  10. Activation of a Ca(2+)-dependent cation conductance with properties of TRPM2 by reactive oxygen species in lens epithelial cells.

    PubMed

    Keckeis, Susanne; Wernecke, Laura; Salchow, Daniel J; Reichhart, Nadine; Strauß, Olaf

    2017-08-01

    Ion channels are crucial for maintenance of ion homeostasis and transparency of the lens. The lens epithelium is the metabolically and electrophysiologically active cell type providing nutrients, ions and water to the lens fiber cells. Ca(2+)-dependent non-selective ion channels seem to play an important role for ion homeostasis. The aim of the study was to identify and characterize Ca(2+)- and reactive oxygen species (ROS)-dependent non-selective cation channels in human lens epithelial cells. RT-PCR revealed gene expression of the Ca(2+)-activated non-selective cation channels TRPC3, TRPM2, TRPM4 and Ano6 in both primary lens epithelial cells and the cell line HLE-B3, whereas TRPM5 mRNA was only found in HLE-B3 cells. Using whole-cell patch-clamp technique, ionomycin evoked non-selective cation currents with linear current-voltage relationship in both cell types. The current was decreased by flufenamic acid (FFA), 2-APB, 9-phenanthrol and miconazole, but insensitive to DIDS, ruthenium red, and intracellularly applied spermine. H2O2 evoked a comparable current, abolished by FFA. TRPM2 protein expression in HLE-B3 cells was confirmed by means of immunocytochemistry and western blot. In summary, we conclude that lens epithelial cells functionally express Ca(2+)- and H2O2-activated non-selective cation channels with properties of TRPM2. Copyright © 2017. Published by Elsevier Ltd.

  11. Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light.

    PubMed

    Padgaonkar, Vanita A; Leverenz, Victor R; Bhat, Aparna V; Pelliccia, Sara E; Giblin, Frank J

    2015-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. © 2014 The American Society of Photobiology.

  12. Cadmium-induced induction of cell death in human lens epithelial cells: implications to smoking associated cataractogenesis.

    PubMed

    Kalariya, Nilesh M; Nair, Bindu; Kalariya, Denish K; Wills, Nancy K; van Kuijk, Frederik J G M

    2010-09-15

    Cadmium is reported to accumulate in human eye tissues suggesting its implication in diverse ocular pathology. Using an in vitro cell culture model we investigated the effects of cadmium on human lens epithelial cells (HLECs) (HLE-B3). We observed cadmium-induced dose- as well as time-dependent decline in HLECs viability which was exacerbated significantly upon reduction of intracellular glutathione levels by buthionine sulfoximine (BSO). There was a dose-dependent significant increase in lactate dehydrogenase (LDH) release from HLECs suggesting cadmium-induced alteration of membrane integrity as well as necrotic cell death. The decline in cell viability was also due to apoptosis of the HLECs as determined by quantifying % apoptotic cells as well as PARP cleavage. Moreover, release of apoptosis inducing factor (AIF) into the cytosol was also detected. Cadmium was also observed to increase oxidative stress, lipid peroxidation and activation of MAPK pathway in HLECs. Antioxidants like N-acetylcysteine (NAC) and alpha-Tocopherol significantly prevented cadmium-induced toxicity in HLECs. Our findings suggest that cadmium-induced elevated oxidative stress as well as activation of MAPK signaling cascade eventually led to cell death of HLECs through apoptosis as well as necrosis. The loss of HLECs by cadmium could possibly explain its implication in cataract development particularly associated with smoking.

  13. Effects of senescent lens epithelial cells on the severity of age-related cortical cataract in humans: A case-control study.

    PubMed

    Fu, Qiuli; Qin, Zhenwei; Yu, Jiexin; Yu, Yinhui; Tang, Qiaomei; Lyu, Danni; Zhang, Lifang; Chen, Zhijian; Yao, Ke

    2016-06-01

    The aging of lens progenitor cell has been repeatedly proposed to play a key role in age-related cataracts (ARCs), but the mechanism is far from being understood. The present study aims to investigate the relationship between aging of lens progenitor/epithelial cells and the 4 subtypes of ARCs in humans.Lens capsules, which were collected from ARC patients during surgery, were divided into 3 groups according to the age of patients (50-60, 60-80, and >80 years). The expressions of lens progenitor cell-related markers Sox2, Abcg2, and Ki67 were first examined in human lens epithelial cells (HLECs) in situ. Then, the percentage of senescent and SA-β-gal HLECs isolated from lens capsules were quantified. Finally, the potential relationships between the percentage of senescent (and SA-β-gal) HLECs and the severity of ARCs were analyzed.Ki67, Sox2, and Abcg2 HLECs in lens capsules were clearly more abundant in young people than in patients older than 50 years, and they were almost absent in patients older than 60 years. The percentage of primary HLECs with aging morphology increased with age, consistent with the results of SA-β-gal primary HLECs. Only cortical cataract classification was found to be strongly related to the percentage of SA-β-gal and senescent HLECs.Our study gave the initial evidence on the dynamical change of lens stem/progenitor cells in human lens capsule with age and suggested that lens progenitor/epithelial cell aging is important in the severity of cortical cataracts.

  14. [Blocking 1800 MHz mobile phone radiation-induced reactive oxygen species production and DNA damage in lens epithelial cells by noise magnetic fields].

    PubMed

    Wu, Wei; Yao, Ke; Wang, Kai-jun; Lu, De-qiang; He, Ji-liang; Xu, Li-hong; Sun, Wen-jun

    2008-01-01

    To investigate whether the exposure to the electromagnetic noise can block reactive oxygen species (ROS) production and DNA damage of lens epithelial cells induced by 1800 MHz mobile phone radiation. The DCFH-DA method and comet assay were used respectively to detect the intracellular ROS and DNA damage of cultured human lens epithelial cells induced by 4 W/kg 1800 MHz mobile phone radiation or/and 2 muT electromagnetic noise for 24 h intermittently. 1800 MHz mobile phone radiation at 4 W/kg for 24 h increased intracellular ROS and DNA damage significantly (P<0.05). However, the ROS level and DNA damage of mobile phone radiation plus noise group were not significant enhanced (P>0.05) as compared to sham exposure group. Electromagnetic noise can block intracellular ROS production and DNA damage of human lens epithelial cells induced by 1800 MHz mobile phone radiation.

  15. αB-crystallin is essential for the TGF-β2-mediated epithelial to mesenchymal transition of lens epithelial cells

    PubMed Central

    Nahomi, Rooban B.; Pantcheva, Mina B.; Nagaraj, Ram H.

    2016-01-01

    Transforming growth factor (TGF)-β2-mediated pathways play a major role in the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) during secondary cataract formation, which is also known as posterior capsule opacification (PCO). Although αB-crystallin is a major protein in LEC, its role in the EMT remains unknown. In a human LEC line (FHL124), TGF-β2 treatment resulted in changes in the EMT-associated proteins at the mRNA and protein levels. This was associated with nuclear localization of αB-crystallin, phosphorylated Smad2 (pSmad2) (S245/250/255), pSmad3 (S423/425), Smad4 and Snail and the binding of αB-crystallin to these transcription factors, all of which were reduced by the down-regulation of αB-crystallin. Expression of the functionally defective R120G mutant of αB-crystallin reduced TGF-β2-induced EMT in LECs of αB-crystallin knockout (KO) mice. Treatment of bovine lens epithelial explants and mouse LEC with TGF-β2 resulted in changes in the EMT-associated proteins at the mRNA and protein levels. This was accompanied by increase in phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) (T202/Y204), p38 MAPK (T180/Y182), protein kinase B (Akt) (S473) and Smad2 when compared with untreated cells. These changes were significantly reduced in αB-crystallin depleted or knocked out LEC. The removal of the fibre cell mass from the lens of wild-type (WT) mice resulted in the up-regulation of EMT-associated genes in the capsule-adherent epithelial cells, which was reduced in the αB-crystallin KO mice. Together, our data show that αB-crystallin plays a central role in the TGF-β2-induced EMT of LEC. αB-Crystallin could be targeted to prevent PCO and pathological fibrosis in other tissues. PMID:26987815

  16. Extremely Low Frequency Magnetic Fields Do Not Induce DNA Damage in Human Lens Epithelial Cells In Vitro.

    PubMed

    Zhu, Kan; Lv, Ye; Cheng, Qian; Hua, Jianing; Zeng, Qunli

    2016-05-01

    Non-ionizing radiations, e.g., radiofrequency electromagnetic fields, could induce DNA damage and oxidative stress in human lens epithelial cells (LECs) which can be early events in cataractogenesis. Extremely low frequency magnetic fields (ELF MF) as another common form of man-made electromagnetic fields has been considered as suspected human carcinogen by International Agency for Research on Cancer (IARC) and become a focus that people play more and more attentions to. This study aimed to determine whether ELF MF can induce DNA damage in cultured human LECs at a relatively low intensity. Human LECs were exposed or sham-exposed to a 50 Hz ELF MF which produced by a well-designed exposure system at the intensity of 0.4 mT. DNA damage in human LECs was examined by the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay and further explored with western blot, flow cytometry, and alkaline comet assay. Immunofluorescence analysis showed that 0.4 mT ELF MF did not significantly increase γH2AX foci formation in human LECs after 2, 6, 12, 24, or 48 hr exposure. No significant differences had been detected in γH2AX expression level between the ELF MF- and sham-exposure groups, while no obvious chromosomal DNA fragmentation was detected by alkaline comet assay after ELF MF exposure. The results indicate an absence of genotoxicity in ELF MF-exposed human epithelial cells and do not support the hypothesis that environmental ELF MF might be causally led to genomic instability via chromosomal damage response processes. Neither short nor long term continuous exposure to 50 Hz ELF MF at 0.4 mT could induce DNA damage in human lens epithelial cells in vitro.

  17. Photochemistry and Photocytotoxicity of Alkaloids from Goldenseal (Hydrastis canadensis L.) 3. Effect on Human Lens and Retinal Pigment Epithelial Cells

    PubMed Central

    Chignell, C.F.; Sik, R.H.; Watson, M.A.; Wielgus, A.R.

    2008-01-01

    The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 μM) and exposed to UVA (5 J/cm2). Under the same conditions palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J/cm2) both berberine (10 μM) and palmatine (10 μM) caused mild DNA damage as determined by the alkaline Comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (λ>400 nm) approximately ten times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged RPE DNA. Infusions of Goldenseal are estimated to contain ∼1 m

  18. Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.

    PubMed

    Albert, Réka; Veréb, Zoltán; Csomós, Krisztián; Moe, Morten C; Johnsen, Erik O; Olstad, Ole Kristoffer; Nicolaissen, Bjørn; Rajnavölgyi, Eva; Fésüs, László; Berta, András; Petrovski, Goran

    2012-01-01

    A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.

  19. Effects of lentiviral short hairpin RNA silencing of Toll-like receptor 4 on the lens epithelial cell line HLEC.

    PubMed

    Yu, H T; Lu, P R

    2016-06-03

    The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.

  20. Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification

    PubMed Central

    Jiang, Yong-Xiang; Liu, Tian-Jing; Yang, Jin; Chen, Yan; Fang, Yan-Wen

    2011-01-01

    Purpose To establish a novel, targeted lentivirus-based HSV-tk (herpes simplex virus thymidine kinase)/GCV (ganciclovir) gene therapy system to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification (PCO) after cataract surgery. Methods An enhanced Cre recombinase (Cre/loxP) system with a lentiviral vector expressing Cre under the control of the lens-specific promoter LEP503 (Lenti-LEP503-HSVtk-Cre [LTKCRE]) was constructed, as well as another lentiviral vector containing a switching unit. The latter vector contains a stuffer sequence encoding EGFP (Lenti-hPGK-Loxp-EGFP-pA-Loxp-HSVtk [PGFPTK]) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-tk) gene, both under the control of the human posphoglycerate kinase (hPGK) promoter. Expression of the downstream gene (HSV-tk) is activated by co-expression of Cre. Human lens epithelial cells (HLECs) or retinal pigmental epithelial cells (RPECs) were co-infected with LTKCRE and PGFPTK. The inhibitory effects on HLECs and RPECs infected by the enhanced specific lentiviral vector combination at the concentration of 20 µg/ml GCV were assayed and compared. Results The specific gene expression of Cre and HSV-tk in HLECs is activated by the LEP503 promoter. LTKCRE and PGFPTK co-infected HLECs, but not RPECs, expressed high levels of the HSV-tk protein. After 96 h of GCV treatment, the percentage of apoptotic HLECs infected by the enhanced specific lentiviral vector combination was 87.23%, whereas that of apoptotic RPECs was only 10.12%. Electron microscopy showed that GCV induced apoptosis and necrosis of the infected HLECs. Conclusions The enhanced specific lentiviral vector combination selectively and effectively expressed HSV-tk in HLECs. A concentration of 20 µg/ml, GCV is effective against the proliferation of HLECs in vitro. This cell-type-specific gene therapy using a Cre/loxP lentivirus system may be a

  1. Development and use of the lens epithelial explant system to study lens differentiation and cataractogenesis.

    PubMed

    West-Mays, Judith A; Pino, Guiseppe; Lovicu, Frank J

    2010-03-01

    Over the last two decades much progress has been made in identifying and characterizing many of the molecules involved in understanding normal lens biology and its pathology. Much of this has been made possible through the establishment and use of the lens epithelial explant system. This simplistic tissue culture model, comprised of a sheet of lens epithelium on its native substratum, has been used effectively to study many cellular processes, including lens epithelial cell proliferation, fiber cell differentiation, cell apoptosis as well as epithelial-to-mesenchymal transformation of cells. In doing so, a number of key growth factors and cytokines, including members of the FGF, Wnt and TGFbeta family have been shown to play essential roles in many of these cellular events. This has led to further studies exploring the signaling pathways downstream of these molecules in the lens, paving the way for the development of a number of in situ models (primarily transgenic mouse lines) to further explore in more detail the nature of these molecular and cellular interactions. To reciprocate, the lens epithelial explant system is increasingly being used to further characterize the nature of many complex phenotypes and pathologies observed in these in situ models, allowing us to selectively isolate and examine the direct impact of an individual molecule on a specific cellular response in lens cells. There is no question that the lens epithelial explant system has served as a powerful tool to further our understanding of lens biology and pathology, and there is no doubt that it will continue to serve in such a capacity, as new developments are realized and putative treatments for aberrant lens cell behavior are to be trialed.

  2. Enhanced cellular uptake and anti-proliferating effect of chitosan hydrochlorides modified genistein loaded NLC on human lens epithelial cells.

    PubMed

    Zhang, Wenji; Liu, Jinlu; Zhang, Qi; Li, Xuedong; Yu, Shihui; Yang, Xinggang; Kong, Jun; Pan, Weisan

    2014-08-25

    This study was attempted to increase the cellular uptake of developed genistein loaded nanostructured lipid carriers (NLC) into human lens epithelial (HLE) cells by chitosan hydrochlorides coatings when applied in post lens capsule (PCO) treatment, and to provide further understanding of the uptake and anti-proliferation mechanisms inside. NLCs were produced using melt-emulsification method and were subsequently coated with chitosan hydrochlorides by adsorption. The uptake of various particle sizes were evaluated and visualized by confocal laser scanning microscopy (CLSM), showing a size-dependent manner. The uptake of NLC was proved to be endocytosed in an energy dependent and clathrin-mediated endocytosis to HLE cells by the decrease in uptake at lower temperature, when pre-saturated by blank NLC and in the presence of NaN3 and sucrose. CH coating improved the uptake percentage of NLC irrespective of the particle size, without influencing the uptake mechanism. Cell apoptosis was tested using PI and Annexin V-FITC/PI staining, followed by flow cytometer analysis. Higher anti-proliferation effect was observed for CH-NLC in inhibiting the growth of HLE cells by causing more apoptosis. Results above indicate that GEN-NLC surface modified by chitosan hydrochlorides could enhance the trans-cellular performance and anti-proliferating effect as PCO therapy.

  3. SiRNA Targeting mTOR Effectively Prevents the Proliferation and Migration of Human Lens Epithelial Cells

    PubMed Central

    Zhang, Chunmei; Liu, Jingjing; Jin, Na; Zhang, Guiming; Xi, Yahui; Liu, Hongling

    2016-01-01

    Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing

  4. Conditional deletion of beta1-integrin from the developing lens leads to loss of the lens epithelial phenotype.

    PubMed

    Simirskii, Vladimir N; Wang, Yan; Duncan, Melinda K

    2007-06-15

    Beta1-integrins are cell surface receptors that participate in sensing the cell's external environment. We used the Cre-lox system to delete beta1-integrin in all lens cells as the lens vesicle transitions into the lens. Adult mice lacking beta1-integrin in the lens are microphthalmic due to apoptosis of the lens epithelium and neonatal disintegration of the lens fibers. The first morphological alterations in beta1-integrin null lenses are seen at 16.5 dpc when the epithelium becomes disorganized and begins to upregulate the fiber cell markers beta- and gamma-crystallins, the transcription factors cMaf and Prox1 and downregulate Pax6 levels demonstrating that beta1-integrin is essential to maintain the lens epithelial phenotype. Furthermore, beta1-integrin null lens epithelial cells upregulate the expression of alpha-smooth muscle actin and nuclear Smad4 and downregulate Smad6 suggesting that beta1-integrin may brake TGFbeta family signaling leading to epithelial-mesenchymal transitions in the lens. In contrast, beta1-integrin null lens epithelial cells show increased E-cadherin immunoreactivity which supports the proposed role of beta1-integrins in mediating complete EMT in response to TGFbeta family members. Thus, beta1-integrin is required to maintain the lens epithelial phenotype and block inappropriate activation of some aspects of the lens fiber cell differentiation program.

  5. Interaction of intraocular lenses with fibronectin and human lens epithelial cells: Effect of chemical composition and aging.

    PubMed

    Tortolano, Lionel; Serrano, Carole; Jubeli, Emile; Saunier, Johanna; Yagoubi, Najet

    2015-12-01

    The aim of this study is to investigate in vitro interactions between hydrophobic acrylate intraocular lenses (IOLs) and their biological environment. The influence of lens chemical composition and aging on fibronectin (FN) adsorption and on IOLs cytotoxicity on human lens epithelial cells was examined. Cytotoxicity of acrylate monomers used in IOLs manufacture was also investigated. Four different IOLs were included in the study: Acrysof(®), Tecnis(®), EnVista(®), and iSert(®). Implants were artificially aged in a xenon arc chamber to simulate 2 years of light exposure. Fibronectin adsorption on IOL surface was quantified using ELISA and correlated to surface roughness determined with AFM. Direct contact cytotoxicity was determined with the MTT assay and cell morphology was observed with light microscopy. Results showed that fibronectin adsorption did not differ significantly among IOLs, whatever their chemical composition. Moreover, aging conditions did not impact fibronectin adsorption. All IOLs were biocompatible even after applying 2-year aging conditions, with cell viability higher than 70%. Five acrylate monomers appeared to be toxic in the range of concentrations tested, but no monomer release from the IOLs could be detected during accelerated 2-year incubation with saline solution. This study did not reveal an influence of chemical composition and aging on protein adsorption and on biocompatibility.

  6. The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness.

    PubMed

    Schonthaler, Helia B; Franz-Odendaal, Tamara A; Hodel, Corinne; Gehring, Ines; Geisler, Robert; Schwarz, Heinz; Neuhauss, Stephan C F; Dahm, Ralf

    2010-04-01

    The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper (bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum(-/-) zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4cM interval on chromosome 7 with the closest markers located at 0.2 and 0cM, respectively. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  7. Source-Dependent Intracellular Distribution of Iron in Lens Epithelial Cells Cultured Under Normoxic and Hypoxic Conditions

    PubMed Central

    Goralska, Małgorzata; Nagar, Steven; Fleisher, Lloyd N.; Mzyk, Philip; McGahan, M. Christine

    2013-01-01

    Purpose. Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of 59Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs). Because lens tissue physiologically functions under low oxygen tension, we also tested effects of hypoxia on iron trafficking. Excess iron, not bound to proteins, can be damaging to cells due to its ability to catalyze formation of reactive oxygen species. Methods. LECs were labeled with 59Fe-Tf or 59FeCl3 under normoxic or hypoxic conditions. Cell lysates were fractioned into mitochondria-rich, nuclei-rich, and cytosolic fractions. Iron uptake and its subcellular distribution were measured by gamma counting. Results. 59Fe accumulation into LECs labeled with 59Fe-Tf was 55-fold lower as compared with that of 59FeCl3. Hypoxia (24 hours) decreased uptake of iron from transferrin but not from FeCl3. More iron from 59FeCl3 was directed to the mitochondria-rich fraction (32.6%–47.7%) compared with 59Fe from transferrin (10.6%–12.6%). The opposite was found for the cytosolic fraction (8.7%–18.3% and 54.2%–46.6 %, respectively). Hypoxia significantly decreased iron accumulation in the mitochondria-rich fraction of LECs labeled with 59Fe-Tf . Conclusions. There are source-dependent differences in iron uptake and trafficking. Uptake and distribution of NTBI are not as strictly regulated as that of iron from transferrin. Excessive exposure to NTBI, which could occur in pathological conditions, may oxidatively damage organelles, particularly mitochondria. PMID:24194187

  8. Source-dependent intracellular distribution of iron in lens epithelial cells cultured under normoxic and hypoxic conditions.

    PubMed

    Goralska, Małgorzata; Nagar, Steven; Fleisher, Lloyd N; Mzyk, Philip; McGahan, M Christine

    2013-11-19

    Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of ⁵⁹Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs). Because lens tissue physiologically functions under low oxygen tension, we also tested effects of hypoxia on iron trafficking. Excess iron, not bound to proteins, can be damaging to cells due to its ability to catalyze formation of reactive oxygen species. LECs were labeled with ⁵⁹Fe-Tf or ⁵⁹FeCl₃ under normoxic or hypoxic conditions. Cell lysates were fractioned into mitochondria-rich, nuclei-rich, and cytosolic fractions. Iron uptake and its subcellular distribution were measured by gamma counting. ⁵⁹Fe accumulation into LECs labeled with ⁵⁹Fe-Tf was 55-fold lower as compared with that of ⁵⁹FeCl₃. Hypoxia (24 hours) decreased uptake of iron from transferrin but not from FeCl₃. More iron from ⁵⁹FeCl₃ was directed to the mitochondria-rich fraction (32.6%-47.7%) compared with ⁵⁹Fe from transferrin (10.6%-12.6%). The opposite was found for the cytosolic fraction (8.7%-18.3% and 54.2%-46.6 %, respectively). Hypoxia significantly decreased iron accumulation in the mitochondria-rich fraction of LECs labeled with ⁵⁹Fe-Tf . There are source-dependent differences in iron uptake and trafficking. Uptake and distribution of NTBI are not as strictly regulated as that of iron from transferrin. Excessive exposure to NTBI, which could occur in pathological conditions, may oxidatively damage organelles, particularly mitochondria.

  9. Spectrum and Range of Oxidative Stress Responses of Human Lens Epithelial Cells to H2O2 Insult

    PubMed Central

    Goswami, Sumanta; Sheets, Nancy L.; Zavadil, Jiři; Chauhan, Bharesh K.; Bottinger, Erwin P.; Reddy, Venkat N.; Kantorow, Marc; Cvekl, Aleš

    2007-01-01

    Purpose Oxidative stress (OS) is believed to be a major contributor to age-related cataract and other age-related diseases. Methods cDNA microarrays were used to identify the spectrum and range of genes with transcript levels that are altered in response to acute H2O2-induced OS in human lens epithelial (HLE) cells. HLE cells were treated with 50 μM H2O2 for 1 hour in the absence of serum, followed by a return to complete medium. RNAs were prepared from treated and untreated cells at 0, 1, 2, and 8 hours after H2O2 treatment. Results The data showed 1171 genes that were significantly up- and downregulated in response to H2O2 treatment. Several functional subcategories of genes were identified, including those encoding DNA repair proteins, antioxidant defense enzymes, molecular chaperones, protein biosynthesis enzymes, and trafficking and degradation proteins. Differential expression of selected genes was confirmed at the level of RNA and/or protein. Many of the identified genes (e.g., glutathione S-transferase [MGST2], thioredoxin reductase β, and peroxiredoxin 2) have been identified as participants in OS responses in the lens and other systems. Some genes induced by OS in the current study (e.g., oxygen regulated protein [ORP150] and heat shock protein [HSP40]) are better known to respond to other forms of stress. Two genes (receptor tyrosine kinase [AXL/ARK] and protein phosphatase 2A) are known to be differentially expressed in cataract. Most of the genes point to a novel pathways associated with OS. Conclusions The present data provide a global perspective on those genes that respond to acute OS, point to novel genes and pathways associated with OS, and set the groundwork for understanding the functions of OS-related genes in lens protection and disease. PMID:12714647

  10. The effect of UVB irradiation on ferritin subunit synthesis, ferritin assembly and Fe metabolism in cultured canine lens epithelial cells.

    PubMed

    Harned, J; Grimes, A M; McGahan, M C

    2003-04-01

    Ferritin is a multimeric protein consisting of heavy and light chains assembled in different tissue-specific ratios, which can protect cells from oxidative stress by storing reactive iron (Fe). Because the lens is constantly exposed to UV irradiation, we studied its effects on ferritin synthesis and Fe metabolism in cultured lens epithelial cells with and without ascorbic acid (Asc). UVB caused a large increase in accumulation of newly synthesized ferritin chains; this increase was additive to that induced by Asc. In contrast to the Asc-induced increase in Fe storage, Fe storage in ferritin was unaltered by UVB. Although UVB increased accumulation of newly synthesized ferritin chains, total ferritin levels were unaltered. In contrast, Asc, which induced a quantitatively similar increase in accumulation of newly synthesized ferritin chains, doubled the total amount of ferritin. Because UVB did not change Fe storage in ferritin or the size of the labile Fe pool, it was hypothesized and then determined that these newly synthesized chains did not assemble into functional holoferritin. Numerous studies detail the effects of various treatments on de novo ferritin synthesis; however, this study provides a cautionary note regarding the conclusions of such studies in the absence of data indicating assembly of functional ferritin molecules.

  11. Parkin elimination of mitochondria is important for maintenance of lens epithelial cell ROS levels and survival upon oxidative stress exposure.

    PubMed

    Brennan, Lisa; Khoury, Josef; Kantorow, Marc

    2017-01-01

    Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to H2O2-oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency.

  12. The effect of catalase amplification on immortal lens epithelial cell lines.

    PubMed

    Yang, Y; Spector, A; Ma, W; Wang, R R; Larsen, K; Kleiman, N J

    1998-12-01

    Utilizing a human beta-actin promoter, a catalase cDNA expression vector was constructed. This construct was used to transfect two immortal cell lines, mouse alpha TN4-1 and rabbit N/N 1003A. The catalase activity was increased about 3.4 fold in the alpha TN4-1 cells and 38 fold in the N/N 1003A cells. Some changes in other enzyme activities were also observed as a result of the transfections. Surprisingly, the ability to degrade H2O2 in the extracellular environment of the cells did not markedly change as a result of the catalase amplification. However, the ability to resist H2O2 stress was dramatically altered. Non-protein thiol (NP-SH) levels, choline uptake and glyceraldehyde phosphate dehydrogenase (GPD) activity were all markedly decreased in the non-transfected cells when they were subjected to 300 microM H2O2. However, in both transfected cell lines, these parameters remained in the normal range during H2O2 stress. The results obtained upon observing aspects of DNA metabolism were more complicated. While on H2O2 stress, non-transfected cell lines showed a marked decrease in thymidine incorporation, only the transfected alpha TN4-1 line remained in the normal range. Thymidine incorporation in transfected rabbit N/N 1003A cells was decreased compared to normal cells. In contrast, studies on single strand DNA breaks indicated that transfected rabbit cells had little damage compared to the significant DNA damage observed in the normal cells. The normal N/N 1003A cells were also much more susceptible to H2O2 induced damage than normal alpha TN4-1 cells, suggesting that the high GSH peroxidase activity observed in the rabbit cells may be detrimental since the low glutathione reductase activity in such cells results in an accelerated depletion of glutathione. The overall results suggest that augmenting lens catalase may prevent cataract development caused by H2O2 stress.

  13. Changes in gap junction organization and decreased coupling during induced apoptosis in lens epithelial and NIH-3T3 cells.

    PubMed

    Theiss, Carsten; Mazur, Antonina; Meller, Karl; Mannherz, Hans Georg

    2007-01-01

    We demonstrate that global induction of apoptosis in primary bovine lens epithelial (LEC) or fibroblastic mouse NIH-3T3 cells by staurosporine, puromycin, cycloheximide, or etoposide is accompanied by a decrease in coupling by gap junctions. Cell coupling as tested by neurobiotin spreading was maintained when the LEC or NIH-3T3 cells were pre-incubated with the pan-caspase inhibitor zVAD or the caspase-3 inhibiting tetrapeptide DEVD. Immunohistochemistry using anti-connexin-43 antibodies showed a reduction of plasma membrane integrated connexin-43 in both cell lines when undergoing apoptosis. Western blotting indicated degradation of connexin-43 that was inhibited by zVAD or DEVD. Cell coupling at single cell level was tested by direct microinjecting into LEC apoptosis-inducing agents of low molecular mass like staurosporine, etoposide and puromycin or the high molecular mass proteins caspase-3 and -8 in activated state. Microinjection of puromycin or etoposide induced apoptotic morphological changes of only the injected cell within 90 or 180 min, but did not affect adjacent cells. In contrast, microinjection of staurosporine led to a rapid induction of apoptosis of the injected and a number of adjacent cells suggesting spreading of staurosporine most probably through gap junction pores held open by dephosphorylation of connexin-43 as verified by immunoblotting and staining using a phospho-serine368-specific anti-connexin-43 antibody. Microinjection of active caspase-8 led after 3 h to morphological apoptotic alterations of only the injected cell, but did not inhibit spreading of co-injected neurobiotin to neighboring cells during the first hour. In contrast, microinjection of active caspase-3-induced apoptosis only of the injected cell after 60 min and rapidly and completely suppressed coupling to neighboring cells.

  14. Lutein and zeaxanthin supplementation reduces H2O2-induced oxidative damage in human lens epithelial cells

    PubMed Central

    Gao, Shasha; Qin, Tingyu; Liu, Zhenzhen; Caceres, Maria Andrea; Ronchi, Carlos F.; Chen, C-Y. Oliver; Yeum, Kyung-jin; Taylor, Allen; Blumberg, Jeffery B.; Liu, Yizhi

    2011-01-01

    Purpose Epidemiological studies suggest that dietary intake of lutein and zeaxanthin is inversely related to the risk for senile cataract. The objectives of this work were to investigate the mechanisms by which these nutrients provide anti-cataract effects. We evaluated their modulation of oxidative damage in human lens epithelial cells (HLEC) and their interaction with intracellular glutathione (GSH). Methods Subconfluent HLEC were pre-incubated with or without 5 µM lutein, zeaxanthin, or α-tocopherol for 48 h and then exposed to 100 µM H2O2 for 1 h. Levels of protein carbonyls in the cells were measured by western-blotting analysis following reaction with 2,4-dinitrophenylhydrazine (DNPH). Levels of malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by an HPLC system. DNA damage was assessed using comet assays. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Results In the absence of H2O2, HLEC had very low levels of protein carbonyl and MDA. Supplementation with lutein, zeaxanthin, or α-tocopherol to the unstressed HLEC had no detectable effects on levels of oxidized proteins and lipid in the cells. Exposure of HLEC to H2O2 significantly increased levels of oxidized proteins, lipid peroxidation, and DNA damage. Pre-incubation with lutein, zeaxanthin, or α-tocopherol dramatically reduced the levels of H2O2 -induced protein carbonyl, MDA, and DNA damage in HLEC. The protective effects of lutein, zeaxanthin, and α-tocopherol against protein oxidation, lipid peroxidation, and DNA damage were comparable. Supplementation with lutein, zeaxanthin, or α-tocopherol increased GSH levels and GSH:GSSG ratio, particularly in response to oxidative stress. Depletion of GSH resulted in significant increase in susceptibility to H2O2-induced cell death. Supplementation with α-tocopherol, but not lutein or zeaxanthin, can partially restore the

  15. Zinc chloride inhibits human lens epithelial cell migration and proliferation involved in TGF-β1 and TNF-α signaling pathways in HLE B-3 cells.

    PubMed

    Du, Yuxiang; Guo, Dadong; Wu, Qiuxin; Liu, Dongmei; Bi, Hongsheng

    2014-06-01

    Zinc is one of the most abundant essential elements in the human body, which is an essential, coenzyme-like component of many enzymes, and is indispensable to their functions. However, high levels of zinc ions can lead to cell damage. In the present study, we explored the effects of high concentrations of zinc chloride (ZnCl2) on lens epithelial cell proliferation and migration and further investigated the effects of different concentrations of ZnCl2 on caspase-9 and caspase-12, transforming growth factor-beta 1 (TGF-β1), and tumor necrosis factor-alpha (TNF-α). We found that ZnCl2 could inhibit human lens epithelial (HLE) B-3 cell migration and induce apoptosis/necrosis. In addition, ZnCl2 can efficiently decrease the expressions of caspase-9 and caspase-12, increase the expression of TNF-α at both gene and protein levels, and thus induces cell death. Taken together, our results indicate that ZnCl2 can inhibit HLE B-3 cell migration and proliferation by decreasing the expression of TGF-β1 and increasing the expression of TNF-α and finally lead to HLE B-3 cell death.

  16. Study of Oxidative Stress in Human Lens Epithelial Cells Exposed to 1.8 GHz Radiofrequency Fields

    PubMed Central

    Ni, Shuang; Yu, Yibo; Zhang, Yidong; Wu, Wei; Lai, Kairan; Yao, Ke

    2013-01-01

    Objectives The aims of the present study were to determine oxidative stress and to explore possible reasons of reactive oxygen species (ROS) increase in human lens epithelial (HLE) B3 cells exposed to low intensity 1.8 GHz radiofrequency fields (RF). Methods The HLE B3 cells were divided into RF exposure and RF sham-exposure groups. The RF exposure intensity was at specific absorption rate (SAR) of 2, 3, or 4 W/kg. The ROS levels were measured by a fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA) assay in the HLE B3 cells exposed to 1.8 GHz RF for 0.5, 1, and 1.5 h. Lipid peroxidation and cellular viability were detected by an MDA test and Cell Counting Kit-8 (CCK-8) assays, respectively, in the HLE B3 cells exposed to 1.8 GHz RF for 6, 12, and 24 h, respectively. The mRNA expression of SOD1, SOD2, CAT, and GPx1 genes and the expression of SOD1, SOD2, CAT, and GPx1 proteins was measured by qRT-PCR and Western blot assays in the HLE B3 cells exposed to 1.8 GHz RF for 1 h. Results The ROS and MDA levels significantly increased (P<0.05) in the RF exposure group and that the cellular viability, mRNA expression of four genes, and expression of four proteins significantly decreased (P<0.05) compared with the RF sham-exposure group. Conclusions Oxidative stress is present in HLE B3 cells exposed to 1.8 GHz low-intensity RF and that the increased production of ROS may be related to down-regulation of four antioxidant enzyme genes induced by RF exposure. PMID:23991100

  17. Overexpression of H- and L-ferritin subunits in lens epithelial cells: Fe metabolism and cellular response to UVB irradiation.

    PubMed

    Goralska, M; Holley, B L; McGahan, M C

    2001-07-01

    To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress. Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA: Fe uptake and incorporation into ferritin was determined by incubating transfected cells with (59)Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation. Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected. Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.

  18. Study of oxidative stress in human lens epithelial cells exposed to 1.8 GHz radiofrequency fields.

    PubMed

    Ni, Shuang; Yu, Yibo; Zhang, Yidong; Wu, Wei; Lai, Kairan; Yao, Ke

    2013-01-01

    The aims of the present study were to determine oxidative stress and to explore possible reasons of reactive oxygen species (ROS) increase in human lens epithelial (HLE) B3 cells exposed to low intensity 1.8 GHz radiofrequency fields (RF). The HLE B3 cells were divided into RF exposure and RF sham-exposure groups. The RF exposure intensity was at specific absorption rate (SAR) of 2, 3, or 4 W/kg. The ROS levels were measured by a fluorescent probe 2'7'-dichlorofluorescin diacetate (DCFH-DA) assay in the HLE B3 cells exposed to 1.8 GHz RF for 0.5, 1, and 1.5 h. Lipid peroxidation and cellular viability were detected by an MDA test and Cell Counting Kit-8 (CCK-8) assays, respectively, in the HLE B3 cells exposed to 1.8 GHz RF for 6, 12, and 24 h, respectively. The mRNA expression of SOD1, SOD2, CAT, and GPx1 genes and the expression of SOD1, SOD2, CAT, and GPx1 proteins was measured by qRT-PCR and Western blot assays in the HLE B3 cells exposed to 1.8 GHz RF for 1 h. The ROS and MDA levels significantly increased (P<0.05) in the RF exposure group and that the cellular viability, mRNA expression of four genes, and expression of four proteins significantly decreased (P<0.05) compared with the RF sham-exposure group. Oxidative stress is present in HLE B3 cells exposed to 1.8 GHz low-intensity RF and that the increased production of ROS may be related to down-regulation of four antioxidant enzyme genes induced by RF exposure.

  19. Expression of transcription factors Slug in the lens epithelial cells undergoing epithelial-mesenchymal transition induced by connective tissue growth factor

    PubMed Central

    Wang, Ying-Na; Qin, Li; Li, Jing-Ming; Chen, Li; Pei, Cheng

    2015-01-01

    AIM To investigate the expression of transcription factors Slug in human lens epithelial cells (HLECs) undergoing epithelial-mesenchymal transition (EMT) induced by connective tissue growth factor (CTGF). METHODS HLECs were treated with CTGF of different concentrations (20, 50 and 100 ng/mL) or without CTGF (control) for 24h. The morphological changes of HLECs were analysed by microscopy. The expression and cellular localization of Slug was evaluated by immumo-fluorescence. Expressions of Slug, E-cadherin and alpha smooth muscle actin (α-SMA) were further determined by Western blot analysis. RESULTS HLECs showed spidle fibrolasts-like characteristics and loosely connected each other after CTGF treatment. The immuno-fluorescence staining indicated that Slug was localized in the nuclei and its expression was induced by CTGF. The relative expressions of Slug protein were 1.64±0.11, 1.96 ±0.03, 3.12 ±0.10, and 4.08±0.14, respectively, in response to control group and treatment with CTGF of 20, 50 and 100 ng/mL (F=443.86, P<0.01). The increased Slug protein levels were correlated well with up-expression of α-SMA (0.78±0.05, 0.85±0.06, 2.17±0.15, 2.86±0.10; F=449.85, P<0.01) and down-expression of E-cadherin (2.50±0.11, 1.79±0.26, 1.05±0.14, 0.63±0.08; F=101.55, P<0.01). CONCLUSION Transcription factor Slug may be involved in EMT of HLECs induced by CTGF in vitro. PMID:26558194

  20. Treatment with retinoic acid and lens epithelial cell-conditioned medium in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

    PubMed

    Chen, Ping; Chen, Jun-Zhao; Shao, Chun-Yi; Li, Chuan-Yin; Zhang, Yi-Dan; Lu, Wen-Juan; Fu, Yao; Gu, Ping; Fan, Xianqun

    2015-02-01

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have extensive self-renewal capacity and the potential to differentiate into all tissue-specific cell lineages, including corneal endothelial cells (CECs). They are a promising prospect for the future of regenerative medicine. The method of derivation of CECs from ESCs and iPSCs, however, remains to be elucidated. In this study, mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development events as a guide. All-trans retinoic acid (RA) treatment during the embryoid body (EB) differentiation step was used to promote neural crest (NC) cell differentiation as first step and was followed by a second induction in CEC- or lens epithelial cell (LEC)-conditioned medium (CM) to ultimately generate CEC-like cells. During the corresponding differentiation stages, NC developmental markers and CEC differentiation markers were detected at the protein level using immunocytochemistry (ICC) and at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). During the first stage, the data indicated that 4 days of treatment with 1 μM RA starting on day 4 of EB formation favored NC cell differentiation and that plating on gelatin-coated plates led to cell migration out of the EBs. The second-stage differentiation results showed that the CM, particularly the LEC-CM, enhanced the yield of polygonal cells with CEC-specific marker expression shown by ICC and RT-qPCR. This study demonstrates that mouse ESCs and iPSCs were induced and expressed CEC differentiation markers when subjected to a two-step inducement process, suggesting that they are a promising resource for corneal endothelium failure replacement therapy in the future.

  1. Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells – Relevance to Capsular Opacification

    PubMed Central

    Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

    2016-01-01

    Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation. PMID:27832099

  2. Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts.

    PubMed

    Palsamy, Periyasamy; Bidasee, Keshore R; Ayaki, Masahiko; Augusteyn, Robert C; Chan, Jefferson Y; Shinohara, Toshimichi

    2014-07-01

    Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of the unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces endoplasmic reticulum stress and activates the unfolded protein response leading to overproduction of reactive oxygen species before human lens epithelial cell death. Methylglyoxal also suppresses Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to overexpression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing shows that human clear lenses (n = 15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n = 21) lose an average of 90% of the 5-methylcytosine regardless of age. Overexpressed Keap1 protein is responsible for decreasing Nrf2 by proteasomal degradation, thereby suppressing Nrf2-dependent stress protection. This study demonstrates for the first time the associations of unfolded protein response activation, Nrf2-dependent antioxidant system failure, and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, the cellular redox balance is altered toward lens oxidation and cataract formation.

  3. Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells

    PubMed Central

    Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

    2008-01-01

    Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). Methods An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay. Results After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by γH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 μT electromagnetic noise could block RF-induced ROS increase and DNA damage. Conclusions DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage. PMID:18509546

  4. Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells.

    PubMed

    Yao, Ke; Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

    2008-05-19

    The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (gammaH2AX) foci formation assay. After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by gammaH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 muT electromagnetic noise could block RF-induced ROS increase and DNA damage. DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage.

  5. Involvement of MsrB1 in the regulation of redox balance and inhibition of peroxynitrite-induced apoptosis in human lens epithelial cells.

    PubMed

    Jia, Yi; Li, Yi; Du, Shaoqing; Huang, Kaixun

    2012-07-01

    Methionine sulfoxide reductases (Msrs) in lens cells are important for the maintenance of lens cell viability and resistance to oxidative stress damage. Peroxynitrite (ONOO(-)), as a strong oxidizing and nitrating agent, occurred in diabetic retinopathy patients and diabetic model animal. In an attempt to shed light on the roles of MsrB1, known as selenoprotein R, in protecting human lens epithelial (HLE) cells against peroxynitrite damage, and contribution of loss of its normal activity to cataract, the influences of MsrB1 gene silencing on peroxynitrite-induced apoptosis in HLE cells were studied. The results showed that both exogenous peroxynitrite and MsrB1 gene silencing by short interfering RNA (siRNA) independently resulted in oxidative stress, endoplasmic reticulum (ER) stress, activation of caspase-3 as well as an increase of apoptosis in HLE cells; moreover, when MsrB1-gene-silenced cells were exposed to 300 μM peroxynitrite, these indexes were further aggravated at the same conditions and DNA strand breaks occurred. The results demonstrate that in HLE cells MsrB1 may play important roles in regulating redox balance and mitigating ER stress as induced by oxidative stress under physiological conditions; MsrB1 may also protect HLE cells against peroxynitrite-induced apoptosis by inhibiting the activation of caspase-3 and oxidative damage of DNA under pathological conditions. Our results imply that loss of its normal activity is likely to contribute to cataract.

  6. Protective effects of Semiaquilegia adoxoides n-butanol extract against hydrogen peroxide-induced oxidative stress in human lens epithelial cells.

    PubMed

    Liang, Bing; Wei, Wei; Wang, Jianta; Zhang, Mingming; Xu, Ran; Wu, Fei; Xiao, Haitao; Tang, Lei

    2016-09-01

    Context Hydrogen peroxide (H2O2)-induced damage in the lens epithelium leads to cell death and cataract. Semiaquilegia adoxoides (DC.) Makino (Ranunculaceae), a folk medicine of Hmong (an ethnic group of China), has been traditionally used to treat cataract; however, the underlying molecular mechanism is yet to be uncovered. Objective This study aimed to investigate whether the n-butanol extract of S. adoxoides (nSA) is effective against the H2O2-induced oxidative stress in human lens epithelial (HLE) cells. Materials and methods Human lens epithelial (SRA 01/04) cells were stimulated by H2O2 (250 μM) in the presence or absence of nSA. The antioxidant effects of nSA were determined in terms of cell viability (MTT assay), apoptosis (AnnexinV/PI staining), radical scavenging capability (various enzymatic assays), loss of mitochondrial membrane potential (Rhodamine 123 staining), expression of apoptotic markers including caspase-3 and caspase-9 and the change of Bcl-2/Bax ratio (western blot) in the HLE cells. Results The results showed that pretreatment of nSA (250, 500 and 1000 μg/mL) markedly reduced H2O2-induced cellular apoptosis and malondialdehyde accumulation, but elevated the activities of total superoxide dismutase, catalase, glutathione peroxidase. Thus, the total antioxidative capability was enhanced upon the nSA treatment meanwhile the loss of mitochondrial membrane potential was prevented. Moreover, nSA at concentrations of 250, 500 and 1000 μg/mL also significantly suppressed the activation of caspase-3 and -9, and increased the Bcl-2/Bax ratio in the HLE cells. Discussion and conclusion Our findings suggested that nSA is a potential prophylactic agent in the prevention of cataractogeneis.

  7. Three-hundred-sixty degree barrier effect of a square-edged and an enhanced-edge intraocular lens on centripetal lens epithelial cell migration Two-year results.

    PubMed

    Vyas, Ashokkumar V; Narendran, Rajesh; Bacon, Peter J; Apple, David J

    2007-01-01

    To study the 360-degree barrier effect of an intraocular lens (IOL) with a square edge at the optic and an enhanced square edge at the optic-haptic junctions (Rayner 570C C-flex) on centripetal migration of lens epithelial cells (LECs) over a 2-year period. Department of Ophthalmology, Scarborough Hospital, Scarborough, United Kingdom. In a prospective study of 40 consecutive eyes, a C-flex IOL was implanted in the bag after phacoemulsification surgery. Eyes with intraoperative complications, requiring additional procedures, without 360-degree overlap of the optic, or with capsule block syndrome were excluded. Follow-up was at 6, 10, 18, and 24 months. At each visit, high-magnification retroillumination digital photographs were taken using a slitlamp-attached digital camera. The barrier effect to LEC migration across the optic edge and the enhanced square edge at the optic-haptic junction was graded as complete (no epithelial pearls or sheet), partial (few epithelial pearls without sheet), and minimal/none (epithelial sheet behind the IOL optic). Twenty-four patients came to the final follow-up at 24 months. Fifteen of these eyes (63%) had a complete barrier effect throughout the 360 degrees of the IOL. Three eyes (13%) had a partial barrier effect throughout the 360 degrees of the IOL. Three eyes had a complete optic barrier effect but a partial optic-haptic junction barrier effect. Three eyes had a partial optic barrier effect but a complete optic-haptic junction barrier effect. No eye had epithelial sheets extending behind the optic at any location. This study showed the barrier effect of the edge design of the C-flex IOL and the efficacy of the enhanced edge in preventing LEC migration at the optic-haptic junction. The enhanced edge was as effective as a sharp square edge in restricting the LEC migration.

  8. Selenoprotein R Protects Human Lens Epithelial Cells against d-Galactose-Induced Apoptosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress

    PubMed Central

    Dai, Jie; Liu, Hongmei; Zhou, Jun; Huang, Kaixun

    2016-01-01

    Selenium is an essential micronutrient for humans. Much of selenium’s beneficial influence on health is attributed to its presence within 25 selenoproteins. Selenoprotein R (SelR), known as methionine sulfoxide reductase B1 (MsrB1), is a selenium-dependent enzyme that, like other Msrs, is required for lens cell viability. In order to investigate the roles of SelR in protecting human lens epithelial (hLE) cells against damage, the influences of SelR gene knockdown on d-galactose-induced apoptosis in hLE cells were studied. The results showed that both d-galactose and SelR gene knockdown by siRNA independently induced oxidative stress. When SelR-gene-silenced hLE cells were exposed to d-galactose, glucose-regulated protein 78 (GRP78) protein level was further increased, mitochondrial membrane potential was significantly decreased and accompanied by a release of mitochondrial cytochrome c. At the same time, the apoptosis cells percentage and the caspase-3 activity were visibly elevated in hLE cells. These results suggested that SelR might protect hLE cell mitochondria and mitigating apoptosis in hLE cells against oxidative stress and endoplasmic reticulum (ER) stress induced by d-galactose, implying that selenium as a micronutrient may play important roles in hLE cells. PMID:26875981

  9. Selenoprotein R Protects Human Lens Epithelial Cells against D-Galactose-Induced Apoptosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress.

    PubMed

    Dai, Jie; Liu, Hongmei; Zhou, Jun; Huang, Kaixun

    2016-02-10

    Selenium is an essential micronutrient for humans. Much of selenium's beneficial influence on health is attributed to its presence within 25 selenoproteins. Selenoprotein R (SelR), known as methionine sulfoxide reductase B1 (MsrB1), is a selenium-dependent enzyme that, like other Msrs, is required for lens cell viability. In order to investigate the roles of SelR in protecting human lens epithelial (hLE) cells against damage, the influences of SelR gene knockdown on d-galactose-induced apoptosis in hLE cells were studied. The results showed that both d-galactose and SelR gene knockdown by siRNA independently induced oxidative stress. When SelR-gene-silenced hLE cells were exposed to d-galactose, glucose-regulated protein 78 (GRP78) protein level was further increased, mitochondrial membrane potential was significantly decreased and accompanied by a release of mitochondrial cytochrome c. At the same time, the apoptosis cells percentage and the caspase-3 activity were visibly elevated in hLE cells. These results suggested that SelR might protect hLE cell mitochondria and mitigating apoptosis in hLE cells against oxidative stress and endoplasmic reticulum (ER) stress induced by d-galactose, implying that selenium as a micronutrient may play important roles in hLE cells.

  10. Variation in cellular glutathione peroxidase activity in lens epithelial cells, transgenics and knockouts does not significantly change the response to H2O2 stress.

    PubMed

    Spector, A; Yang, Y; Ho, Y S; Magnenat, J L; Wang, R R; Ma, W; Li, W C

    1996-05-01

    This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H2O2 in lens preparations. Rabbit (N/N1003A) and normal and GSHPx-1 transfected mouse (alpha TN4-1) lens epithelial cell lines and normal and GSHPx-1 transgenic and knockout mouse lenses were utilized. GSHPx-1 activity in the cell lines was increased from two-fold to about four-fold, in the lenses from transgenics more than four-fold and the lenses from knockouts had less than 3% of normal GSHPx-1 activity. The transgenic and knockout mice as well as their lenses appeared normal for up to 3 to 4 months, the longest period of observation. The preparations were subjected to oxidative stress by placing them either in a medium containing 120 or 300 microM H2O2 or utilizing photochemical stress where the H2O2 levels normally rise to about 100 microM over a few hours in the presence of a normal lens. With all preparations, it was found that either markedly increasing or eliminating GSHPx-1 activity had only a small effect on the system's ability to metabolize H2O2, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase (GSSG Red) and 3-aminotriazole (3-AT), an inhibitor of catalase, also had little effect. However, the addition of both inhibitors caused a marked decrease in H2O2 degradation. Examination of the distribution of GSHPx-1 in the lens indicated that the activity per milligram of protein was evenly distributed between the epithelium and the remainder of the lens in the normal lens and was about 1.7-fold greater in the epithelium of transgenic lenses than in the remainder of the lens. Surprisingly, the distribution of GSSG Red was quite different with eight- to ten-fold more activity in the epithelium. Catalase was also found to be concentrated in the epithelium. With H2O2 exposure, a rapid loss of non-protein thiol (NP-thiol) was found in cell cultures and in the epithelia of cultured lenses. However, the remainder of the lens showed little change

  11. MicroRNA-34a promoting apoptosis of human lens epithelial cells through down-regulation of B-cell lymphoma-2 and silent information regulator

    PubMed Central

    Li, Qing-Lan; Zhang, Hong-Yang; Qin, Yong-Jie; Meng, Qian-Li; Yao, Xiao-Lei; Guo, Hai-Ke

    2016-01-01

    AIM To investigate the role of microRNA-34a (miR-34a) in the induction of apoptosis of human lens epithelial (HLE-B3) cells. METHODS The apoptosis of HLE-B3 cells was detected by Annexin V-PE apoptosis detection kit after the treatment with 200 µmol/L H2O2 for 24h and lentiviral miR-34a vector transfection. The expression of miR-34a in the cells was quantified by quantitative real time polymerase chain reaction (qRT-PCR) in response to H2O2 exposure and the vector transfection. The effects of overexpression of miR-34a on the expression of B-cell lymphoma-2 (Bcl-2) and silent information regulator 1 (SIRT1) was determined by qRT-PCR and Western blot. RESULTS The expression of miR-34a was up-regulated by the treatment of H2O2 in HLE-B3 cells. The increased expression of miR-34a is accompanied with the cell apoptosis. Consistence with the H2O2 exposure, ectopic overexpression of miR-34a in HLE-B3 cells promoted cells apoptosis. Importantly the anti-apoptosis factors Bcl-2 and SIRT1 were reduced significantly by up-regulation of miR-34a in HLE-B3 cells. CONCLUSION MiR-34a promotes the apoptosis of HLE-B3 cells by down-regulating Bcl-2 and SIRT1, suggesting that miR-34a may involve in the pathogenesis of cataract formation and targeting miR-34a may be a potentially therapeutic approach for treatment of cataract. PMID:27990356

  12. Effects of white light-emitting diode (LED) light exposure with different correlated color temperatures (CCTs) on human lens epithelial cells in culture.

    PubMed

    Xie, Chen; Li, Xiuyi; Tong, Jianping; Gu, Yangshun; Shen, Ye

    2014-01-01

    Cataract is the major cause for legal blindness in the world. Oxidative stress on the lens epithelial cells (hLECs) is the most important factor in cataract formation. Cumulative light-exposure from widely used light-emitting diodes (LEDs) may pose a potential oxidative threat to the lens epithelium, due to the high-energy blue light component in the white-light emission from diodes. In the interest of perfecting biosafety standards for LED domestic lighting, this study analyzed the photobiological effect of white LED light with different correlated color temperatures (CCTs) on cultured hLECs. The hLECs were cultured and cumulatively exposed to multichromatic white LED light with CCTs of 2954, 5624, and 7378 K. Cell viability of hLECs was measured by Cell Counting Kit-8 (CCK-8) assay. DNA damage was determined by alkaline comet assay. Intracellular reactive oxygen species (ROS) generation, cell cycle, and apoptosis were quantified by flow cytometry. Compared with 2954 and 5624 K LED light, LED light having a CCT of 7378 K caused overproduction of intracellular ROS and severe DNA damage, which triggered G2 /M arrest and apoptosis. These results indicate that white LEDs with a high CCT could cause significant photobiological damage to hLECs.

  13. Difference in Phototoxicity of Cyclodextrin Complexed Fullerene [(γ-CyD)2/C60] and Its Aggregated Derivatives toward Human Lens Epithelial Cells

    PubMed Central

    Zhao, Baozhong; He, Yu-Ying; Chignell, Colin F.; Yin, Jun-Jie; Andley, Usha; Roberts, Joan E.

    2009-01-01

    The water-soluble fullerene derivative γ-cyclodextrin bicapped C60 [(γ-CyD)2/C60, CDF0] has several clinical applications, including use as a drug carrier to bypass the blood ocular barriers or a photosensitizer to treat tumors in photodynamic therapy. We have assessed the potential ocular toxicity of (γ-CyD)2/C60 and its aggregated derivatives induced by UVA and visible light in vitro in human lens epithelial cells (HLE B-3). Cell viability using the MTS assay demonstrated that 2 μM (γ-CyD)2/C60 was highly phototoxic to HLE B-3 cells with UVA irradiation, while no effect was observed in the presence of visible light or when maintained in the dark. In contrast, the aggregated derivative (CDF150) showed neither cytotoxicity nor any phototoxic effect even at 30 μM with either UVA or visible light irradiation. In lens cells treated with (γ-CyD)2/C60, phototoxicity was manifested as apoptosis. Singlet oxygen production measurement using the EPR/TEMP trapping technique determined that (γ-CyD)2/C60 (CDF0) efficiently produced singlet oxygen. The rate of singlet oxygen production decreased with increased aggregation, with no production by the fully aggregated sample formed after 150 min of heating (CDF150). UVA irradiation of HLE B-3 in the presence of (γ-CyD)2/C60 resulted in a significant rise in intracellular protein-derived peroxides. The singlet oxygen quenchers sodium azide and histidine each significantly protected lens cells against (γ-CyD)2/C60 photodamage, but lutein and Trolox (vitamin E) did not. Clearly, singlet oxygen is an important intermediate in the phototoxicity of monomeric (γ-CyD)2/fullerene. Our results also demonstrate that UVA-blocking sunglasses can limit the ocular phototoxicity of this nanomaterial, while nontoxic endogenous antioxidants like lutein or Trolox cannot provide adequate protection. PMID:19281132

  14. Glucocorticoid receptors take part in the apoptotic process of human lens epithelial cells, but the glucocorticoid receptor antagonist RU486 does not rescue the cells fully.

    PubMed

    Wang, Lin; Zhao, Wencheng; Leng, Fei; Ge, Jinying; Bu, Zhigao; Zhang, Yi; Liu, Ping

    2011-06-01

    To identify an agent with specific activity against human lens epithelial cells (HLECs), we confirmed the presence of glucocorticoid receptors (GRs) and GR-α genes and evaluated whether GRs have a relationship with the apoptotic process in cultured HLECs. We also determined whether the inhibitor RU486 could rescue the cells from apoptosis when the HLECs were exposed to dexamethasone (Dex), a steroid, in 4 concentrations for 4 periods, or were co-treated with the antagonist RU486. We found that Dex, which has been used as a medical agent for a long time, resulted in increased expression of GRE-luciferase, the GR-α gene and GR-protein and, in contrast, decreased the viability of HLECs. The expression of Bax protein was increased in an earlier stage in contrast to the expression of Bcl-2 protein, which was increased in a later stage. Caspase-3 activity was significantly increased under lower concentrations of Dex in the last stage. The nuclear morphology of HLECs showed an obvious apoptotic phenomenon under greater concentrations of Dex in the last stage. However, RU486, a GR antagonist, could partially inhibit GR and Bax expressions and the expression of caspase-3 was increased so that there was not a decrease in the ratio of apoptotic cells and an increase in the viability of HLECs. Our data showed that GRs had a partial relationship to the apoptotic process of HLECs when exposed to Dex and RU486 did not rescue the cells fully. Because of its toxicity, RU486 did not provide a therapeutic benefit in a glucocorticoid induced cataract (GIC) for the in vitro model, however, its activity and pathway targeting should still be studied further with appropriate drug combinations.

  15. The lens equator: a platform for molecular machinery that regulates the switch from cell proliferation to differentiation in the vertebrate lens.

    PubMed

    Mochizuki, Toshiaki; Masai, Ichiro

    2014-06-01

    The vertebrate lens is a transparent, spheroidal tissue, located in the anterior region of the eye that focuses visual images on the retina. During development, surface ectoderm associated with the neural retina invaginates to form the lens vesicle. Cells in the posterior half of the lens vesicle differentiate into primary lens fiber cells, which form the lens fiber core, while cells in the anterior half maintain a proliferative state as a monolayer lens epithelium. After formation of the primary fiber core, lens epithelial cells start to differentiate into lens fiber cells at the interface between the lens epithelium and the primary lens fiber core, which is called the equator. Differentiating lens fiber cells elongate and cover the old lens fiber core, resulting in growth of the lens during development. Thus, lens fiber differentiation is spatially regulated and the equator functions as a platform that regulates the switch from cell proliferation to cell differentiation. Since the 1970s, the mechanism underlying lens fiber cell differentiation has been intensively studied, and several regulatory factors that regulate lens fiber cell differentiation have been identified. In this review, we focus on the lens equator, where these regulatory factors crosstalk and cooperate to regulate lens fiber differentiation. Normally, lens epithelial cells must pass through the equator to start lens fiber differentiation. However, there are reports that when the lens epithelium structure is collapsed, lens fiber cell differentiation occurs without passing the equator. We also discuss a possible mechanism that represses lens fiber cell differentiation in lens epithelium.

  16. Heme oxygenase-1 (HO-1) protects human lens epithelial cells (SRA01/04) against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis.

    PubMed

    Ma, Tianju; Chen, Tingjun; Li, Peng; Ye, Zi; Zhai, Wei; Jia, Liang; Chen, Wenqian; Sun, Ang; Huang, Yang; Wei, Shihui; Li, Zhaohui

    2016-05-01

    This study aimed to investigate the protective role of heme oxygenase-1 (HO-1) in H2O2-induced oxidative stress and apoptosis in human lens epithelial cells (hLEC; SRA01/04). SRA01/04 cells were exposed to a hydrogen peroxide (H2O2) concentration gradient and inducers of HO-1 such as cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP), respectively. In addition, an RNA silencing experiment was conducted to investigate the HO-1 function in this study. A Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot and ELISA were used to detect the level of HO-1 expression. In our study, hLECs were exposed to 400 μM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with 10μΜ CoPP or 10μΜ ZnPP, respectively. Double immunofluorescence staining was used for cell identification and the qualitative expression of HO-1. Expression of HO-1 was monitored using Western blot and ELISA. Intracellular reactive oxygen species (ROS) were detected by flow cytometry analyses; commercial enzymatic kits were used to measure the levels of glutathione (GSH), as well as superoxide dismutase (SOD). The proportion of cell apoptosis was quantified by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The expression of caspase family (-8, -3) proteins was measured by Western blot analysis. HO-1 significantly restored the cell viability under H2O2 injury via reducing the generation of ROS and increasing the levels of SOD and GSH activity. Moreover, HO-1 also inhibited H2O2-induced caspase-8 and caspase-3 proteins, thus significantly reducing the apoptosis of SRA01/04. An RNA silencing experiment demonstrated the increased resistance of LECs to oxidative stress specifically for increased levels of HO-1. These findings suggested that HO-1 protects human lens epithelial cells from H2O2-induced oxidant stress by upregulating antioxidant enzyme activity, reducing ROS generation, and thus inhibiting caspase family

  17. The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract.

    PubMed

    Gosak, Marko; Markovič, Rene; Fajmut, Aleš; Marhl, Marko; Hawlina, Marko; Andjelić, Sofija

    2015-01-01

    In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of

  18. The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract

    PubMed Central

    Gosak, Marko; Markovič, Rene; Fajmut, Aleš; Marhl, Marko; Hawlina, Marko; Andjelić, Sofija

    2015-01-01

    In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of

  19. Mitochondrial "movement" and lens optics following oxidative stress from UV-B irradiation: cultured bovine lenses and human retinal pigment epithelial cells (ARPE-19) as examples.

    PubMed

    Bantseev, Vladimir; Youn, Hyun-Yi

    2006-12-01

    Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta

  20. Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis.

    PubMed

    Maddala, Rupalatha; Nagendran, Tharkika; Lang, Richard A; Morozov, Alexei; Rao, Ponugoti V

    2015-10-01

    Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.

  1. Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis

    PubMed Central

    Maddala, Rupalatha; Nagendran, Tharkika; Lang, Richard A.; Morozov, Alexei; Rao, Ponugoti V.

    2015-01-01

    Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis. PMID:26212757

  2. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  3. Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo.

    PubMed

    Kim, Junghyun; Kim, Ohn Soon; Kim, Chan-Sik; Sohn, Eunjin; Jo, Kyuhyung; Kim, Jin Sook

    2012-02-29

    The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor- kappaB (NF-κB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-oneweek- old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-κB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal- treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-κB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-κB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-κB-dependent and pro-apoptotic.

  4. Acetyl-l-carnitine prevents homocysteine-induced suppression of Nrf2/Keap1 mediated antioxidation in human lens epithelial cells.

    PubMed

    Yang, Shui-Ping; Yang, Xiu-Zhen; Cao, Guo-Ping

    2015-07-01

    Previous studies have revealed that high levels of serum homocysteine (Hcy) are closely associated with the development of juvenile and age-related cataracts. An increased concentration of Hcy is likely to induce gene specific demethylation in DNA promoter regions. The aim of the present study was to prevent this demethylation by administering acetyl-l-carnitine (ALCAR) to human lens epithelial cells (HLECs). Different concentrations of Hcy were used to treat HLECs for 3, 6, 12 and 24 h and the findings were used to determine the optimum dose to induce endoplasmic reticulum (ER) stress. Similarly, the concentration of ALCAR was standardized. The production of reactive oxygen species (ROS) and the percentage of cells undergoing cell death were measured. The levels of antioxidants, ER stress-associated proteins, mRNA levels of nuclear factor erythroid-2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) and promoter DNA methylation of the Keap1 gene were also assessed. Hcy was observed to induce ER stress, produce ROS and lead to cell death. However, administration of ALCAR prevented these effects to a significant degree. Additionally, western blot analysis revealed that ALCAR increased the levels of antioxidant proteins, including catalase, superoxide dismutase, glutathione peroxidase, Nrf2, Keap1 and glutathione. Similarly, the reverse transcription-quantitative polymerase chain reaction experiments on Nrf2 and Keap1, as well as the bisulfite genomic DNA sequencing analysis revealed a preventive effect of ALCAR against Hcy-induced ER stress. The ER stress-induced activation of the unfolded protein response is responsible for demethylation of Keap1 promoter DNA to activate the expression of the Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. This decrease in Nrf2 activity represses the transcription of numerous antioxidant enzyme genes and alters the redox-balance towards lens oxidation. However, treatment

  5. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  6. Negative regulation of TGFβ-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists.

    PubMed

    Zhao, Guannan; Wojciechowski, Magdalena C; Jee, Seonah; Boros, Jessica; McAvoy, John W; Lovicu, Frank J

    2015-03-01

    An eclectic range of ocular growth factors with differing actions are present within the aqueous and vitreous humors that bathe the lens. Growth factors that exert their actions via receptor tyrosine kinases (RTKs), such as FGF, play a normal regulatory role in lens; whereas other factors, such as TGFβ, can lead to an epithelial to mesenchymal transition (EMT) that underlies several forms of cataract. The respective downstream intracellular signaling pathways of these factors are in turn tightly regulated. One level of negative regulation is thought to be through RTK-antagonists, namely, Sprouty (Spry), Sef and Spred that are all expressed in the lens. In this study, we tested these different negative regulators and compared their ability to block TGFβ-induced EMT in rat lens epithelial cells. Spred expression within the rodent eye was confirmed using RT-PCR, western blotting and immunofluorescence. Rat lens epithelial explants were used to examine the morphological changes associated with TGFβ-induced EMT over 3 days of culture, as well as α-smooth muscle actin (α-sma) immunolabeling. Cells in lens epithelial explants were transfected with either a reporter (EGFP) vector (pLXSG), or with plasmids also coding for different RTK-antagonists (i.e. pLSXG-Spry1, pLSXG-Spry2, pLXSG-Sef, pLSXG-Spred1, pLSXG-Spred2, pLSXG-Spred3), before treating with TGFβ for up to 3 days. The percentages of transfected cells that underwent TGFβ-induced morphological changes consistent with an EMT were determined using cell counts and validated with a paired two-tailed t-test. Explants transfected with pLXSG demonstrated a distinct transition in cell morphology after TGFβ treatment, with ∼60% of the cells undergoing fibrotic-like cell elongation. This percentage was significantly reduced in cells overexpressing the different antagonists, indicative of a block in lens EMT. Of the antagonists tested under these in vitro conditions, Spred1 was the most potent demonstrating the

  7. Interaction of AR and iNOS in lens epithelial cell: A new pathogenesis and potential therapeutic targets of diabetic cataract.

    PubMed

    Li, Xue; Liu, Wenping; Huang, Xinduo; Xiong, Jianping; Wei, Xiaoyong

    2017-02-01

    Although there is significant interest in revealing the role of aldose reductase (AR) and inducible nitric oxide synthase (iNOS) in diabetic cataract (DC), the interaction of AR and iNOS remains unknown. The aim of this study is to investigate the pathogenesis mechanisms and explore as a new potential therapeutic targets for DC. This study investigated the interaction of AR-iNOS through the methods of enzyme kinetics, molecular docking and molecular dynamics simulation, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). The IC50 of AR for inhibition of iNOS activity is 0.04 μM, and the IC50 of iNOS for inhibition of AR activity is 0.042 μM through enzyme kinetics; the interface showed that ARG99 on AR and GLU317 on iNOS played the key roles in the interaction of AR-iNOS predicted by molecular docking and molecular dynamics simulation. Co-immunoprecipitation of protein complexes in human lens epithelial cell (HLEC) demonstrated that AR could association with iNOS in cell; and the interaction distance of AR-iNOS was 6.50 ± 0.22 nm detected by FRET. This study exhibited a direct inhibition interaction between AR and iNOS in HLECs. It is the first report of inhibition interaction between AR and iNOS, suggesting a new pathophysiological mechanism and providing a new insight into the therapeutic mechanism of DC. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. c-myc Regulates Cell Proliferation during Lens Development

    PubMed Central

    Gomes, Anielle L.; Rodrigues, Paulo M. G.; Martins, Rodrigo A. P.

    2014-01-01

    Myc protooncogenes play important roles in the regulation of cell proliferation, growth, differentiation and survival during development. In various developing organs, c-myc has been shown to control the expression of cell cycle regulators and its misregulated expression is detected in many human tumors. Here, we show that c-myc gene (Myc) is highly expressed in developing mouse lens. Targeted deletion of c-myc gene from head surface ectoderm dramatically impaired ocular organogenesis, resulting in severe microphtalmia, defective anterior segment development, formation of a lens stalk and/or aphakia. In particular, lenses lacking c-myc presented thinner epithelial cell layer and growth impairment that was detectable soon after its inactivation. Defective development of c-myc-null lens was not caused by increased cell death of lens progenitor cells. Instead, c-myc loss reduced cell proliferation, what was associated with an ectopic expression of Prox1 and p27Kip1 proteins within epithelial cells. Interestingly, a sharp decrease in the expression of the forkhead box transcription factor Foxe3 was also observed following c-myc inactivation. These data represent the first description of the physiological roles played by a Myc family member in mouse lens development. Our findings support the conclusion that c-myc regulates the proliferation of lens epithelial cells in vivo and may, directly or indirectly, modulate the expression of classical cell cycle regulators in developing mouse lens. PMID:24503550

  9. Primary cultures of embryonic chick lens cells as a model system to study lens gap junctions and fiber cell differentiation.

    PubMed

    Musil, Linda S

    2012-07-01

    A major limitation in lens gap junction research has been the lack of experimentally tractable ex vivo systems to study the formation and regulation of fiber-type gap junctions. Although immortalized lens-derived cell lines are amenable to both gene transfection and siRNA-mediated knockdown, to our knowledge none are capable of undergoing appreciable epithelial-to-fiber differentiation. Lens central epithelial explants have the converse limitation. A key advance in the field was the development of a primary embryonic chick lens cell culture system by Drs. Sue Menko and Ross Johnson. Unlike central epithelial explants, these cultures also include cells from the peripheral (preequatorial and equatorial) epithelium, which is the most physiologically relevant population for the study of fiber-type gap junction formation. We have modified the Menko/Johnson system and refer to our cultures as dissociated cell-derived monolayer cultures (DCDMLs). We culture DCDMLs without serum to mimic the avascular lens environment and on laminin, the major matrix component of the lens capsule. Here, I review the features of the DCDML system and how we have used it to study lens gap junctions and fiber cell differentiation. Our results demonstrate the power of DCDMLs to generate new findings germane to the mammalian lens and how these cultures can be exploited to conduct experiments that would be impossible, prohibitively expensive and/or difficult to interpret using transgenic animals in vivo.

  10. Glutathione and catalase suppress TGFβ-induced cataract-related changes in cultured rat lenses and lens epithelial explants

    PubMed Central

    Chamberlain, Coral G.; Cerra, Anna

    2009-01-01

    Purpose The damaging effects of oxidative stress and transforming growth factor-β (TGFβ)-induced transdifferentiation of lens epithelial cells have both been implicated independently in the etiology of cataract. The aim of this study was to investigate whether the presence of antioxidant systems in the lens influences the ability of lens epithelial cells to respond to TGFβ. Methods Whole lenses from young rats were cultured with or without TGFβ in the presence or absence of reduced glutathione (GSH). Lens epithelial explants from weanling rats were used to investigate the effects of GSH and catalase on TGFβ-induced cataract-related changes. Lenses were monitored for opacification for three to four days, photographed, and then processed for routine histology. Explants were assessed by phase contrast microscopy, enzyme-linked immunosorbent assay (ELISA) of α-smooth muscle actin (αSMA), and/or immunolocalization of αSMA and Pax6, markers for transdifferentiation and normal lens epithelial phenotype, respectively. Results In cultured lenses, GSH strongly suppressed TGFβ-induced opacification and subcapsular plaque formation. In explants, both GSH and catalase suppressed changes typically associated with TGFβ-induced transdifferentiation including wrinkling of the lens capsule, cell-surface blebbing, apoptotic cell loss, induction of αSMA, and loss of Pax6 expression. Conclusions This study suggests that antioxidant systems present in the normal lens, which protect the epithelium against the damaging effects of reactive oxygen species, may also serve to protect it against the potentially cataractogenic effects of TGFβ. Taken together with other recent studies, it also raises the possibility that TGFβ may induce cataract-related changes in lens epithelial cells via release of hydrogen peroxide. PMID:19421408

  11. Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

    PubMed

    Yamben, Idella F; Rachel, Rivka A; Shatadal, Shalini; Copeland, Neal G; Jenkins, Nancy A; Warming, Soren; Griep, Anne E

    2013-12-01

    The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.

  12. Quercetin inhibited epithelial mesenchymal transition in diabetic rats, high-glucose-cultured lens, and SRA01/04 cells through transforming growth factor-β2/phosphoinositide 3-kinase/Akt pathway.

    PubMed

    Du, Lei; Hao, Meng; Li, Chengcheng; Wu, Wenya; Wang, Wenwen; Ma, Zhongxuan; Yang, Tingting; Zhang, Nan; Isaac, Adelusi Temitope; Zhu, Xia; Sun, Ying; Lu, Qian; Yin, Xiaoxing

    2017-09-05

    Diabetic cataract (DC), an identified life-threatening secondary complication of diabetes mellitus, has proven to be a dilemma because of its multifactorial caused and progression. An increasing number of studies have shown that in addition to the maillard reaction, enhanced polyol pathway, and oxidative insults, epithelial mesenchymal transition (EMT) is related to the prevalence of DC. Quercetin, a classic flavonoid with multiple pharmacological effects has been reported to possess therapeutic efficacy in the management and treatment of this disease. However, the mechanism underlying its therapeutic efficacy in EMT of lens epithelial cells (SRA01/04) and contribution to resolving DC remains a mystery. Therefore, in this study, we investigated the effects of quercetin on EMT of SRA01/04 and high-glucose (HG)-induced lens opacity accompanied by lens fibrosis induced by type-1 diabetes. Furthermore, we sought to clarify the specific mechanisms underlying these effects. At week 14 after streptozotocin (STZ) intraperitoneal administration, diabetic rats showed lens opacity accompanied with diminished antioxidant function, enhanced polyol pathway activity, and non-enzymatic glycation. Western blotting confirmed EMT in rat SRA01/04 cells with significantly increased α-smooth muscle actin (α-SMA) and decreased E-cadherin expressions. Treatment of the lens with quercetin ameliorated the oxidative stress, inhibited aldose reductase (AR) activation, reduced advanced glycation end product (AGE) production, and finally suppressed EMT in the early stages. Our in vitro results showed that high-glucose activated the transforming growth factor-β2/phosphoinositide 3-kinase/protein kinase B (TGF-β2/PI3K/Akt) signalling and EMT in SRA01/04 cells. Further, induced oxidative stress, activation of aldose reductase, and accumulation of advanced glycation end products were also involved in this process. Quercetin was potent enough to effectively ameliorate the high glucose (HG

  13. rs78378222 polymorphism in the 3'-untranslated region of TP53 contributes to development of age-associated cataracts by modifying microRNA-125b-induced apoptosis of lens epithelial cells.

    PubMed

    Zhao, Yang; Li, Xiao; Zhu, Siquan

    2016-09-01

    MicroRNAs (miRNAs) negatively regulate the expression of the target genes by binding to 'seed sequences' in the 3'‑untranslated region (3'‑UTR) mRNA transcripts, and the variants within or nearby 'seed sequences' may compromise or enhance miRNA/mRNA interaction leading to either 'loss‑of‑function' or 'gain‑of‑function' effects. Cataracts are the leading cause of blindness worldwide and are characterized by progressive aggregation and precipitation of lens proteins, and the development of age‑related cataracts is associated with dysregulated cellular activities of lens epithelial cells. Luciferase assays and online miRNA databases were used to validate that tumor protein p53 (TP53) is the target gene of miR‑125b. Furthermore, reverse transcription‑quantitative polymerase chain reaction and western blotting were conducted to detect expression levels of miR‑125b and TP53 in different groups of cells transfected with miR‑125b mimics or inhibitors. In addition, flow cytometry analysis and the MTT assay were conducted to detect the effects of miR‑125b on apoptosis and cell viability. The current study demonstrated that the rs78378222 polymorphism minor allele introduces a novel potential miR‑125b binding site in the TP53 3'‑UTR with a consecutive 8‑bp perfect match, creating a 'gain‑of‑function' variant and affecting the regulation of TP53 expression. A luciferase assay demonstrated that transfection of lens epithelial cells with wild type TP53 3'‑UTR significantly reduced the luciferase activity of the miR‑125b overexpressing cells compared with scramble controls. In addition, the luciferase activity of miR‑125b overexpressing cells transfected with the construct containing the rs78378222 polymorphism minor allele was also reduced compared with cells transfected with the wild type 3'‑UTR. Furthermore, it was demonstrated that the expression level of miR‑125 was comparable in epithelial cells from patients with age

  14. The impact of lens care solutions on corneal epithelial changes during daily silicone hydrogel contact lens wear as measured by in vivo confocal microscopy.

    PubMed

    Zhang, Xiaolin; Marchetti, Christine; Lee, Jessica; Sun, Yan; Debanne, Sara; Jiang, Ying; Kern, Jami; Harrod, Mark; Benetz, Beth Ann; Pearlman, Eric; Szczotka-Flynn, Loretta

    2017-02-01

    To assess corneal epithelial microstructure via confocal microscopy and determine if cellular changes are associated with lens care solutions during daily wear of silicone hydrogel contact lenses. Corneal in vivo confocal microscopy with the Nidek ConfoScan4 was performed at baseline and after 5 months of lotrafilcon A daily contact lens wear. Enrolled participants were randomized to use either a polyhexamethylene biguanide (PHMB) preserved multipurpose care solution (MPS) or a peroxide based solution system. Lens and storage case bioburden were assessed with aerobic culture methods. Univariate and multivariable analyses were done to evaluate the association between solution use, or solution-related clinical covariates, and morphologic differences (hyper-reflectivity) in the superficial epithelial cells and epithelial basal cell density. Data on 139 participants were available for analysis of superficial epithelial cells while data on 92 participants were available for epithelial basal cell density. Five months after randomization to the solution groups, 33% of participants had visible hyper-reflective cells. More participants using MPS had ≥1 hyper-reflective cells compared to peroxide users at 5 months (44% vs. 22%; p=0.006). Similarly at 5 months, more participants with solution-induced corneal staining (SICS) had ≥1 hyper-reflective cells compared to non-SICS participants (57% vs. 29%; p=0.010). The adjusted odds ratios (ORs) for risk of presenting with hyper-reflective cells in MPS users or SICS participants was 2.7 (95% CI; 1.27-5.65) and 3.4 (95% CI; 1.29-8.97), respectively. Basal cell density decreased by over 350 cells/mm(2) over time (about 6%) in participants who had substantial bioburden on their lenses or in their storage case. The confocal microscope can detect epithelial cellular changes in vivo during contact lens wear. Hyper-reflective superficial epithelial cells are associated with a PHMB preserved solution and decreases in basal epithelial

  15. Pax6 is essential for lens fiber cell differentiation.

    PubMed

    Shaham, Ohad; Smith, April N; Robinson, Michael L; Taketo, Makoto M; Lang, Richard A; Ashery-Padan, Ruth

    2009-08-01

    The developing ocular lens provides an excellent model system with which to study the intrinsic and extrinsic cues governing cell differentiation. Although the transcription factors Pax6 and Sox2 have been shown to be essential for lens induction, their later roles during lens fiber differentiation remain largely unknown. Using Cre/loxP mutagenesis, we somatically inactivated Pax6 and Sox2 in the developing mouse lens during differentiation of the secondary lens fibers and explored the regulatory interactions of these two intrinsic factors with the canonical Wnt pathway. Analysis of the Pax6-deficient lenses revealed a requirement for Pax6 in cell cycle exit and differentiation into lens fiber cells. In addition, Pax6 disruption led to apoptosis of lens epithelial cells. We show that Pax6 regulates the Wnt antagonist Sfrp2 in the lens, and that Sox2 expression is upregulated in the Pax6-deficient lenses. However, our study demonstrates that the failure of differentiation following loss of Pax6 is independent of beta-catenin signaling or Sox2 activity. This study reveals that Pax6 is pivotal for initiation of the lens fiber differentiation program in the mammalian eye.

  16. N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis.

    PubMed

    Logan, Caitlin M; Rajakaruna, Suren; Bowen, Caitlin; Radice, Glenn L; Robinson, Michael L; Menko, A Sue

    2017-08-01

    Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Comparative transcriptome analysis of epithelial and fiber cells in newborn mouse lenses with RNA sequencing

    PubMed Central

    Hoang, Thanh V.; Kumar, Praveen Kumar Raj; Sutharzan, Sreeskandarajan; Tsonis, Panagiotis A.; Liang, Chun

    2014-01-01

    Purpose The ocular lens contains only two cell types: epithelial cells and fiber cells. The epithelial cells lining the anterior hemisphere have the capacity to continuously proliferate and differentiate into lens fiber cells that make up the large proportion of the lens mass. To understand the transcriptional changes that take place during the differentiation process, high-throughput RNA-Seq of newborn mouse lens epithelial cells and lens fiber cells was conducted to comprehensively compare the transcriptomes of these two cell types. Methods RNA from three biologic replicate samples of epithelial and fiber cells from newborn FVB/N mouse lenses was isolated and sequenced to yield more than 24 million reads per sample. Sequence reads that passed quality filtering were mapped to the reference genome using Genomic Short-read Nucleotide Alignment Program (GSNAP). Transcript abundance and differential gene expression were estimated using the Cufflinks and DESeq packages, respectively. Gene Ontology enrichment was analyzed using GOseq. RNA-Seq results were compared with previously published microarray data. The differential expression of several biologically important genes was confirmed using reverse transcription (RT)-quantitative PCR (qPCR). Results Here, we present the first application of RNA-Seq to understand the transcriptional changes underlying the differentiation of epithelial cells into fiber cells in the newborn mouse lens. In total, 6,022 protein-coding genes exhibited differential expression between lens epithelial cells and lens fiber cells. To our knowledge, this is the first study identifying the expression of 254 long intergenic non-coding RNAs (lincRNAs) in the lens, of which 86 lincRNAs displayed differential expression between the two cell types. We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data. Gene Ontology analysis showed that genes

  18. Lung Epithelial Progenitor Cells

    PubMed Central

    Rawlins, Emma L.

    2008-01-01

    The current enthusiasm for stem cell research stems from the hope that damaged or diseased tissues may one day be repaired through the manipulation of endogenous or exogenous stem cells. The postnatal human respiratory system is highly accessible and provides unique opportunities for the application of such techniques. Several putative adult lung epithelial stem cells have been identified in the mouse model system. However, their in vivo capabilities to contribute to different lineages, and their control mechanisms, remain unclear. If stem cell–based therapies are to be successful in the lung, it is vitally important that we understand the normal behavior of adult lung stem cells, and how this is regulated. Lung embryonic progenitor cells are much better defined and characterized than their adult counterparts. Moreover, experiments on a variety of developing tissues are beginning to uncover general mechanisms by which embryonic progenitors influence final organ size and structure. This provides a framework for the study of lung embryonic progenitor cells, facilitating experimental design and interpretation. A similar approach to investigating adult lung stem cells could produce rapid advances in the field. PMID:18684716

  19. Protective Effect of D-Limonene against Oxidative Stress-Induced Cell Damage in Human Lens Epithelial Cells via the p38 Pathway

    PubMed Central

    Bai, Jie; Zheng, Yi; Wang, Gang; Liu, Ping

    2016-01-01

    Oxidative stress, as mediated by ROS, is a significant factor in initiating the development of age-associated cataracts; D-limonene is a common natural terpene with powerful antioxidative properties which occurs naturally in a wide variety of living organisms. It has been shown to have antioxidant effect; we found that D-limonene can effectively prevent the oxidative damage caused by H2O2 and propose that the main mechanism underlying the inhibitory effects of D-limonene is the inhibition of HLECs apoptosis. In the present study, we used confocal-fluorescence microscopy, flow cytometry analysis, Hoechst staining, H2DCFDA staining, transmission electron microscopy, and immunoblot analysis; the results revealed that slightly higher concentrations of D-limonene (125–1800 μM) reduced the H2O2-induced ROS generation and inhibited the H2O2-induced caspase-3 and caspase-9 activation and decreased the Bcl-2/Bax ratio. Furthermore, it inhibited H2O2-induced p38 MAPK phosphorylation. Thus, we conclude that D-limonene could effectively protect HLECs from H2O2-induced oxidative stress and that its antioxidative effect is significant, thereby increasing the cell survival rate. PMID:26682012

  20. [Pathological study on effects of preservative-free 1% lidocaine on lens epithelial cells of patients with age-related cataract].

    PubMed

    Guan, Huai-jin; Duan, Hai-xia; Wu, Jian; Zhu, Rong-rong

    2008-12-01

    To assess whether preservative-free 1% lidocaine is capable of destroying the LECs in age-related cataract (ARC) in order to provide scientific basis for pursuing safe and effective drugs to eliminate LECs in cataract surgery. Lens anterior capsule (LAC) specimens were collected from 75 patients (82 eyes) with age-related cataract (ARC), including forty males (44 eyes) and thirty-five females (38 eyes). The age range was 41 - 85 years, the mean age was 67.97 years. There were 34 cortical cataracts, 22 nuclear cataracts and 26 subcapsular cataracts. Capsule specimens were divided into 4 groups: balanced salt solution (BSS) group I and group II (exposed to BSS for 1 minute), lidocaine group (exposed to preservative-free 1% lidocaine for 1 minute) and the control group. Specimens were stained with trypan blue and alizarin red. Photomicrographs of each capsule were taken to observe the viability of LECs and to count the number of necrosis LECs. The pathologic changes of LECs were evaluated by histological methods (11 LAC, 22 pieces) as well as transmission and scanning electron microscopes (5 LAC, 10 pieces). In the control and BSS group I (23 LAC), one half of each capsule specimen was used for the control group and the other half was used for BSS group I. In lidocaine group and BSS group II (43 LAC), one half of each capsule specimen was used for lidocaine group and the other half was used for BSS group II. The rate of necrosis LECs of the anterior capsules in the control group and BSS group I was (56.19 +/- 2.71)% and (57.23 +/- 1.98)%, respectively. The rate of necrosis LECs of the capsules in lidocaine group and BSS group II was (99.86 +/- 8.22)% and (57.64 +/- 7.00)%, respectively. Matching t-test showed that the rate of necrosis LECs in lidocaine group was greater than that in the BSS group II (t = 27.6781, P = 0.0000). There was no significant difference in the number of necrosis LECs between the control group and BSS group I (t = 2.0693, P = 0.0505). There was

  1. A method for determining cell number in the undisturbed epithelium of the mouse lens

    PubMed Central

    Shi, Yanrong

    2010-01-01

    The anterior face of the mouse lens is covered by a layer of epithelial cells. The epithelial cells serve a barrier function at the lens surface and as a progenitor population from which lens fiber cells, the predominant cell type of the lens, are derived. Decreased epithelial cell density is commonly observed during aging and cataract formation in humans and animal models and may contribute directly to tissue opacification. However, the loss of cells from the epithelium is often not easy to quantify, in part because the cells are arrayed across a near-spherical surface and, as a consequence, are difficult to image and count. Here, we describe a technique for determining epithelial cell number in the undisturbed lens of the mouse, a popular cataract model. The method utilizes orthographic projections of confocal images collected from the anterior and equatorial regions of the lens. The overlapping projections are brought into register using the unique distribution of proliferating cells as fiduciary points. Cell counts are performed using a computer-assisted method. This approach offers several advantages over flat-mount methods employed previously. PMID:21139698

  2. Bone Morphogenetic Protein-7 Suppresses TGFβ2-Induced Epithelial-Mesenchymal Transition in the Lens: Implications for Cataract Prevention

    PubMed Central

    Shu, Daisy Y.; Wojciechowski, Magdalena C.; Lovicu, Frank J.

    2017-01-01

    Purpose Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-β (TGFβ) superfamily, TGFβ and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFβ is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFβ. We examine the modulating effect of BMP-7 on TGFβ-induced EMT in LECs. Methods Rat lens epithelial explants were treated exogenously with TGFβ2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id). Results Transforming growth factor-β2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFβ2-induced α-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and β-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFβ2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFβ2-induced pSmad2/3. Treatment with TGFβ2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression. Conclusions Exogenous administration of BMP-7 abrogated TGFβ2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFβ- and BMP-7–associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention. PMID:28152139

  3. Integrins and epithelial cell polarity.

    PubMed

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.

  4. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

  5. Epithelial cells as alternative human biomatrices for comet assay.

    PubMed

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  6. Sef is a negative regulator of fiber cell differentiation in the ocular lens.

    PubMed

    Newitt, Peter; Boros, Jessica; Madakashira, Bhavani P; Robinson, Michael L; Reneker, Lixing W; McAvoy, John W; Lovicu, Frank J

    2010-07-01

    Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.

  7. Lens stem cells may reside outside the lens capsule: an hypothesis.

    PubMed

    Remington, Susann G; Meyer, Rita A

    2007-06-08

    In this paper, we consider the ocular lens in the context of contemporary developments in biological ideas. We attempt to reconcile lens biology with stem cell concepts and a dearth of lens tumors.Historically, the lens has been viewed as a closed system, in which cells at the periphery of the lens epithelium differentiate into fiber cells. Theoretical considerations led us to question whether the intracapsular lens is indeed self-contained. Since stem cells generate tumors and the lens does not naturally develop tumors, we reasoned that lens stem cells may not be present within the capsule. We hypothesize that lens stem cells reside outside the lens capsule, in the nearby ciliary body. Our ideas challenge the existing lens biology paradigm. We begin our discussion with lens background information, in order to describe our lens stem cell hypothesis in the context of published data. Then we present the ciliary body as a possible source for lens stem cells, and conclude by comparing the ocular lens with the corneal epithelium.

  8. Modulation of lens cell adhesion molecules by particle beams

    NASA Technical Reports Server (NTRS)

    McNamara, M. P.; Bjornstad, K. A.; Chang, P. Y.; Chou, W.; Lockett, S. J.; Blakely, E. A.

    2001-01-01

    Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast

  9. Modulation of lens cell adhesion molecules by particle beams

    NASA Technical Reports Server (NTRS)

    McNamara, M. P.; Bjornstad, K. A.; Chang, P. Y.; Chou, W.; Lockett, S. J.; Blakely, E. A.

    2001-01-01

    Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast

  10. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  11. Down-Regulation of MicroRNA-133b Suppresses Apoptosis of Lens Epithelial Cell by Up-Regulating BCL2L2 in Age-Related Cataracts

    PubMed Central

    Zhang, Feng; Meng, Weizhe; Tong, Bin

    2016-01-01

    Background MicroRNA-133b (miR-133b) has been reported to be involved in many diseases, including ovarian cancer and osteosarcoma. Accumulating evidence suggests that miR-133b plays important roles in human disease. In this study, we aimed to investigate the molecular mechanism, including the potential regulator and signaling pathways, of BCL2L2. Material/Methods We first searched the online miRNA database (www.mirdb.org) using the “seed sequence” located within the 3′-UTR of the target gene, and then performed luciferase assay to test the regulatory relationship between miR-133b and BCL2L2. Western blot and real-time PCR were used to determine the expression of BCL2L2 in human samples or cells treated with miRNA mimics or inhibitors. Flow cytometry was conducted to evaluate the apoptosis status of the cells. Results We validated BCL2L2 to be the direct gene using a luciferase reporter assay. We also conducted real-time PCR and Western blot analyses to study the mRNA and protein expression level of BCL2L2 among different groups (control: n=29, cataract: n=33) or cells treated with scramble control, miR-133b mimics, BCL2L2 siRNA, and miR-133b inhibitors, and identified the negative regulatory relationship between miR-133b and BCL2L2. We also conducted experiments to investigate the influence of miR-133b and BCL2L2 on the viability and apoptosis of cells. The results showed that miR-133b positively interfered with the viability of cells, while BCL2L2 negatively interfered with the viability of cells, and that miR-133b inhibited apoptosis while BCL2L2 accelerated apoptosis. Conclusions BCL2L2 was the virtual target of miR-133b, and we found a negative regulatory relationship between miR-133b and BCL2L2. MiR-133b and BCL2L2 interfered with the viability and apoptosis of cells. PMID:27802259

  12. Effects of UV-A radiation on lens epithelial NaK-ATPase in organ culture.

    PubMed

    Dovrat, A; Weinreb, O

    1999-06-01

    To investigate the mechanisms involved in the damage caused by UV-A irradiation at 365 nm on the eye lens. Bovine lenses obtained from animals 1 to 5 years of age were placed in specially designed organ culture chambers for preincubation. Twenty-four hours later, the lenses were irradiated by 33 J/cm2 UV-A at 365 nm. During irradiation, the lenses were oriented in the culture so that the anterior surface faced the incident UV-A radiation source. After irradiation, lens optical quality was monitored throughout the 8 days of the culture period, and lens samples were taken for analysis of NaK-ATPase activity. Lens optics and NaK-ATPase activity were affected by irradiation of 33 J/cm2. The effects on lens epithelial NaK-ATPase activity were stronger at the equators than at the center. The damage to the activity at the center was reversible, as the lens optically recovered from the LW-A damage. Lens NaK-ATPase activity can recover from damage caused by UV-A at 365 nm. When the lenses received irradiation of 33 J/cm2, NaK-ATPase activity recovered from the damage during the culture period only at the center and not at the equators of the epithelium.

  13. Autophagy and mitophagy participate in ocular lens organelle degradation.

    PubMed

    Costello, M Joseph; Brennan, Lisa A; Basu, Subharsee; Chauss, Daniel; Mohamed, Ashik; Gilliland, Kurt O; Johnsen, Sönke; Menko, Sue; Kantorow, Marc

    2013-11-01

    The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Corneal Cell Adhesion to Contact Lens Hydrogel Materials Enhanced via Tear Film Protein Deposition

    PubMed Central

    Elkins, Claire M.; Qi, Qin M.; Fuller, Gerald G.

    2014-01-01

    Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo. PMID:25144576

  15. Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

    PubMed

    Elkins, Claire M; Qi, Qin M; Fuller, Gerald G

    2014-01-01

    Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

  16. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  17. Lens regeneration using endogenous stem cells with gain of visual function.

    PubMed

    Lin, Haotian; Ouyang, Hong; Zhu, Jie; Huang, Shan; Liu, Zhenzhen; Chen, Shuyi; Cao, Guiqun; Li, Gen; Signer, Robert A J; Xu, Yanxin; Chung, Christopher; Zhang, Ying; Lin, Danni; Patel, Sherrina; Wu, Frances; Cai, Huimin; Hou, Jiayi; Wen, Cindy; Jafari, Maryam; Liu, Xialin; Luo, Lixia; Zhu, Jin; Qiu, Austin; Hou, Rui; Chen, Baoxin; Chen, Jiangna; Granet, David; Heichel, Christopher; Shang, Fu; Li, Xuri; Krawczyk, Michal; Skowronska-Krawczyk, Dorota; Wang, Yujuan; Shi, William; Chen, Daniel; Zhong, Zheng; Zhong, Sheng; Zhang, Liangfang; Chen, Shaochen; Morrison, Sean J; Maas, Richard L; Zhang, Kang; Liu, Yizhi

    2016-03-17

    The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

  18. Herpes Simplex epithelial keratitis associated with daily disposable contact lens wear.

    PubMed

    Hamroush, Ahmed; Welch, James

    2014-06-01

    To report a case of epithelial Herpes Simplex keratitis in a patient wearing daily disposable contact lenses. Case report. A 30-year-old female contact lens wearer presented to the emergency clinic with a painful, red left eye associated with an acute reduction of vision over 48 h. On examination, confluent dendritic ulcers were present on the cornea. Neither pertinent ocular nor medical history was obtained to explain such a dramatic clinical presentation. Contact lens wear was the only risk factor identified, perhaps resulting in deviation of the immune response at the ocular surface, with consequent extensive dendritic ulceration. Copyright © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  19. Pseudodendritic fungal epithelial keratitis in an extended wear contact lens user.

    PubMed

    Knape, Robert M; Motamarry, Silus P; Sakhalkar, Monali V; Tuli, Sonal S; Driebe, William T

    2011-01-01

    Pseudodendritic keratitis in a contact lens wearer is generally associated with acanthamoeba keratitis. We report a case of isolated pseudodendritic fungal epithelial keratitis that occurred in an extended wear contact lens user. A 48-year-old woman was evaluated in our clinic for a 36-hour history of left eye pain. She wore extended wear soft contact lenses and frequently rinsed her eyes with tap water. Her left cornea had a paracentral 3-mm area of epithelium with raised ridges in a pseudodendritic pattern. The underlying corneal stroma was normal. A therapeutic and diagnostic corneal scraping of the lesion was performed and sent for Gomori methenamine silver (GMS) staining. The clinical concern was for epithelial acanthamoeba keratitis. The GMS staining revealed septate fungal hyphae within sheets of corneal epithelium. The patient was started on frequent alternating natamycin (5%) and amphotericin B (0.15%) antifungal eyedrops and exhibited a rapid clinical response. Her keratitis completely resolved, and her vision returned to her baseline of 20/25. Corneal fungal cultures showed no growth. Our case is an extremely unusual presentation of fungal keratitis, which rarely presents as a pseudodendritic epithelial keratitis. There are two previous similar case reports initially misdiagnosed as acanthamoeba keratitis. Clinicians should be aware that isolated fungal epithelial keratitis can present as a distinct entity and should be considered in the differential diagnosis of pseudodendritic keratitis. The GMS staining is an excellent diagnostic test in a patient presenting with pseudodendritic keratitis because it allows rapid diagnosis of acanthamoeba and fungal infections.

  20. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  1. Tropomodulin 1 Constrains Fiber Cell Geometry during Elongation and Maturation in the Lens Cortex

    PubMed Central

    Nowak, Roberta B.

    2012-01-01

    Lens fiber cells exhibit a high degree of hexagonal packing geometry, determined partly by tropomodulin 1 (Tmod1), which stabilizes the spectrin-actin network on lens fiber cell membranes. To ascertain whether Tmod1 is required during epithelial cell differentiation to fiber cells or during fiber cell elongation and maturation, the authors quantified the extent of fiber cell disorder in the Tmod1-null lens and determined locations of disorder by confocal microscopy and computational image analysis. First, nearest neighbor analysis of fiber cell geometry in Tmod1-null lenses showed that disorder is confined to focal patches. Second, differentiating epithelial cells at the equator aligned into ordered meridional rows in Tmod1-null lenses, with disordered patches first observed in elongating fiber cells. Third, as fiber cells were displaced inward in Tmod1-null lenses, total disordered area increased due to increased sizes (but not numbers) of individual disordered patches. The authors conclude that Tmod1 is required first to coordinate fiber cell shapes and interactions during tip migration and elongation and second to stabilize ordered fiber cell geometry during maturation in the lens cortex. An unstable spectrin-actin network without Tmod1 may result in imbalanced forces along membranes, leading to fiber cell rearrangements during elongation, followed by propagation of disorder as fiber cells mature. PMID:22473940

  2. Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells.

    PubMed

    Yasukawa, Atsushi; Hosoki, Koa; Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C

    2013-01-01

    Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

  3. Imaging of dense cell cultures by multiwavelength lens-free video microscopy.

    PubMed

    Allier, C; Morel, S; Vincent, R; Ghenim, L; Navarro, F; Menneteau, M; Bordy, T; Hervé, L; Cioni, O; Gidrol, X; Usson, Y; Dinten, J-M

    2017-02-27

    They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm(2) ) over a large field of view of 29.4 mm(2) . Combined with the compactness of the system which fits altogether inside an incubator, lens-free microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry.

  4. Disassembly of the lens fiber cell nucleus to create a clear lens: the p27 descent

    USDA-ARS?s Scientific Manuscript database

    The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFC...

  5. A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells.

    PubMed

    Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

    2012-08-01

    In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals.

  6. A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

    PubMed Central

    Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

    2012-01-01

    In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

  7. Equarin is involved in cell adhesion by means of heparan sulfate proteoglycan during lens development.

    PubMed

    Song, Xiaohong; Sato, Yuya; Sekiguchi, Kiyotoshi; Tanaka, Hideaki; Ohta, Kunimasa

    2013-01-01

    Adhesion molecules are known to be instructive for both development and differentiation. During lens differentiation, epithelial cells undergo vertical elongation, with the anterior and posterior tips of the elongating fiber cells sliding along the epithelium and capsule, respectively. These cellular processes are highly coordinated through cell adhesive interactions, actin cytoskeletal reorganization and contractile force generation. Alterations in extracellular matrix composition that interfere with these interactions can lead to defects that alter tissue morphogenesis and the state of differentiation. We have demonstrated that Equarin, which is a secreted molecule expressed in the equator region of the lens, plays an important role in chick lens fiber differentiation through fibroblast growth factor signaling. Here, we explored the function of Equarin in chick lens cell adhesion. Equarin protein was expressed in the extracellular region of lens differentiating cells. We found that Equarin promoted lens cell adhesion through heparan sulfate proteoglycan. By biochemical analysis, we found that Equarin directly binds syndecan-3, which displayed a similar expression pattern to Equarin. Overexpression of Equarin resulted in altered actin localization. Equarin is involved in cell adhesion during fiber differentiation and development. Copyright © 2012 Wiley Periodicals, Inc.

  8. Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium.

    PubMed

    Shahidullah, M; Mandal, A; Delamere, N A

    2015-11-01

    The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 μM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 μM). Similar changes were observed in response to fiber damage caused by injection of 5 μl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to

  9. ERK1/2 signaling is required for the initiation but not progression of TGFβ-induced lens epithelial to mesenchymal transition (EMT).

    PubMed

    Wojciechowski, Magdalena C; Mahmutovic, Leila; Shu, Daisy Y; Lovicu, Frank J

    2017-06-01

    Transforming Growth Factor Beta (TGFβ) potently induces lens epithelial to mesenchymal transition (EMT). The resultant mesenchymal cells resemble those found in plaques of human forms of subcapsular cataract. Smad signaling has long been implicated as the sole driving force of TGFβ-mediated activity. Rat lens epithelial explants were used to examine the role of the Smad-independent signaling, namely the MAPK/ERK1/2 signaling pathway, in the initiation and progression of TGFβ-induced EMT. Phase contrast microscopy was used to observe the morphological changes associated with TGFβ-induced EMT in this model, including cell elongation, cell membrane blebbing, cell loss as indicated by the area of bare capsule and capsular wrinkling. The levels of Smad2, Smad2/3 and ERK1/2 phosphorylation measured using western blotting confirmed that the addition of UO126 was sufficient in blocking all TGFβ-induced ERK1/2 activation, as well as reducing Smad signaling at 18 h. Immunofluorescent labeling and further western blotting confirmed that TGFβ-induced EMT was associated with an increase in α-smooth muscle actin (α-SMA) and a reduction of E-cadherin at cell borders. Pre-treatment with UO126 was effective at blocking the TGFβ-induced EMT, as evidenced by a reduction of α-SMA expression and protein labeling, E-cadherin labeling at cell borders, and a reduction of cell loss, cell elongation and capsular wrinkling. Post-treatment with UO126 at 2 and 6 h after TGFβ addition was also effective at blocking EMT while post-treatment with UO126 at 24 and 48 h was not sufficient in hampering TGFβ-induced EMT. Our data implicates ERK1/2 signaling in the initiation but not the progression of TGFβ-induced EMT in rat lens epithelial cells. The tight regulation of intracellular signaling pathways such as ERK1/2 are required for the maintenance of lens epithelial cell integrity and hence tissue transparency. A greater understanding of the molecular mechanisms that drive the

  10. Integrin αVβ5-mediated Removal of Apoptotic Cell Debris by the Eye Lens and Its Inhibition by UV Light Exposure.

    PubMed

    Chauss, Daniel; Brennan, Lisa A; Bakina, Olga; Kantorow, Marc

    2015-12-18

    Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin αVβ5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVβ5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVβ5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the αVβ5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVβ5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.

  11. Biomarkers of oxidative stress and cataract. Novel drug delivery therapeutic strategies targeting telomere reduction and the expression of telomerase activity in the lens epithelial cells with N-acetylcarnosine lubricant eye drops: anti-cataract which helps to prevent and treat cataracts in the eyes of dogs and other animals.

    PubMed

    Babizhayev, Mark A; Yegorov, Yegor E

    2014-01-01

    Cataracts in small animals are shown to be at least partially caused by oxidative damage to lens epithelial cells (LECs) and the internal lens; biomarkers of oxidative stress in the lens are considered as general biomarkers for life expectancy in the canine and other animals. Telomeres lengths and expressed telomerase activity in canine LECs may serve as important monitors of oxidative damage in normal LECs with documented higher levels of telomerase activity in cataractous LECs during cells' lifespan. Loss of functional telomere length below a critical threshold in LECs of canines during the effect of UV and chronic oxidative stress or metabolic failure, can activate programs leading to LEC senescence or death. Telomerase is induced in LECs of canines at critical stages of cataractogenesis initiation and exposure to oxidative stress through the involvement of catalytically active prooxidant transition metal (iron) ions. This work documents that transition metal ions (such as, ferrous ions- catalytic oxidants) might induce premature senescence in LECs of canines, telomere shortening with increased telomerase activity as adaptive response to UV light, oxidative and metabolic stresses. The therapeutic treatment with 1% N-acetylcarnosine (NAC) prodrug delivery is beneficial for prevention and dissolution of ripe cataracts in canines. This biological activity is based on the findings of ferroxidase activity pertinent to the dipeptide carnosine released ophthalmically from NAC prodrug of L-carnosine, stabilizing properties of carnosine on biological membranes based on the ability of the imidazole-containing dipeptides to interact with lipid peroxidation products and reactive oxygen species (ROS), to prevent membrane damage and delute the associated with membrane fragements protein aggregates. The advent of therapeutic treatment of cataracts in canines with N-acetylcarnosine lubricant eye drops through targeting the prevention of loss of functional telomere length below

  12. Telomere Attrition in Human Lens Epithelial Cells Associated with Oxidative Stress Provide a New Therapeutic Target for the Treatment, Dissolving and Prevention of Cataract with N-Acetylcarnosine Lubricant Eye Drops. Kinetic, Pharmacological and Activity-Dependent Separation of Therapeutic Targeting: Transcorneal Penetration and Delivery of L-Carnosine in the Aqueous Humor and Hormone-Like Hypothalamic Antiaging Effects of the Instilled Ophthalmic Drug Through a Safe Eye Medication Technique.

    PubMed

    Babizhayev, Mark A; Yegorov, Yegor E

    2016-01-01

    Visual impairment broadly impacts the ability of affected people to maintain their function and to remain independent during their daily occupations as they grow older. Visual impairment affects survival of older patients, quality of life, can affect a person's self-ranking of health, may be associated with social and functional decline, use of community support services, depression, falls, nursing home placement, and increased mortality. It has been hypothesized that senile cataract may serve as a marker for generalised tissue aging, since structural changes occurring in the proteins of the lens during cataract formation are similar to those which occur elsewhere as part of the aging process. The published analysis revealed a strong age-dependent relationship between undergoing cataract surgery and subsequent mortality. Nuclear opacity, particularly severe nuclear opacity, and mixed opacities with nuclear were significant predictors of mortality independent of body mass index, comorbid conditions, smoking, age, race, and sex. The lens opacity status is considered as an independent predictor of 2-year mortality, an association that could not be explained by potential confounders. Telomeres have become important biomarkers for aging as well as for oxidative stress-related disease. The lens epithelium is especially vulnerable to oxidative stress. Oxidative damage to the cuboidal epithelial cells on the anterior surface of the lens mediated by reactive oxygen species and phospholipid hydroperoxides can precede and contribute to human lens cataract formation. The erosion and shortening of telomeres in human lens epithelial cells in the lack of telomerase activity has been recognized as a primary cause of premature lens senescence phenotype that trigger human cataractogenesis. In this study we aimed to be focused on research defining the mechanisms that underlie linkages among telomere attrition in human lens epithelial cells associated with oxidative stress, biology of

  13. Keratin cytoskeletons in epithelial cells of internal organs

    PubMed Central

    Sun, Tung-Tien; Shih, Chiaho; Green, Howard

    1979-01-01

    An antiserum against human epidermal keratins was used to detect keratins in frozen sections of various rabbit and human tissues by indirect immunofluorescence. Strong staining was observed in all stratified squamous epithelia (epidermis, cornea, conjunctiva, tongue, esophagus, vagina, and anus), in epidermal appendages (hair follicle, sebaceous gland, ductal and myoepithelial cells of sweat glands), as well as in Hassall's corpuscles of the thymus, indicating that all contain abundant keratins. No staining by the antiserum was observed in fibroblasts, muscle of any type, cartilage, blood vessel, nerve tissue, iris or lens epithelium, or the glomerular or tubular cells of the kidney. In contrast, the antiserum stained the cells of most epithelia of the intestinal tract, urinary tract (urethra, bladder, ureter, collecting ducts of kidney), female genital tract (cervix, cervical glands, uterus, and oviduct), and respiratory tract (trachea and bronchi). Epithelial cells of the fine ductal system in the pancreas and submaxillary gland also stained well. When primary cultures of epithelial cells derived from bladder, intestine, kidney, and trachea were grown on glass coverslips and stained with anti-keratin, fiber networks similar to those of cultured keratinocytes were observed. These results show that keratins constitute a cytoskeleton in epithelial cells of diverse morphology and embryological origin. The stability of keratin filaments probably confers the structural strength necessary for cells covering a free surface. Keratin staining can be used to obtain information about the origin of cell lines. Images PMID:111242

  14. Patterning Bacterial Communities on Epithelial Cells

    PubMed Central

    Dwidar, Mohammed; Leung, Brendan M.; Yaguchi, Toshiyuki; Takayama, Shuichi; Mitchell, Robert J.

    2013-01-01

    Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv) gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions. PMID:23785519

  15. Epithelial TRPV1 Signaling Accelerates Gingival Epithelial Cell Proliferation

    PubMed Central

    Takahashi, N.; Matsuda, Y.; Yamada, H.; Tabeta, K.; Nakajima, T.; Murakami, S.; Yamazaki, K.

    2014-01-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca2+ levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  16. Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells.

    PubMed

    Boswell, Bruce A; Musil, Linda S

    2015-07-01

    Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency--fiber cell differentiation and gap junction-mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function.

  17. Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye

    PubMed Central

    Murphy, Taylor R.; Vihtelic, Thomas S.; Ile, Kristina E.; Watson, Corey T.; Willer, Gregory B.; Gregg, Ronald G.; Bankaitis, Vytas A.; Hyde, David R.

    2011-01-01

    The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol--inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the cdipt-encoded PI synthase protein phenocopied the cdiptlop/lop mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of in vitro transcribed, wild-type cdipt mRNA into 1-4 cell stage cdiptlop/lop embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a cdipt retroviral-insertion allele, cdipthi559, exhibited similar lens and retinal abnormalities and failed to complement the cdiptlop mutant phenotype. To determine the initial cellular defects associated with the cdipt mutant, we examined homozygous cdipthi559/hi559 mutants prior to gross lens opacification at 6 dpf. The cdipthi559/hi559 mutants first exhibited photoreceptor layer disruption and photoreceptor cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. RT-PCR revealed that the cdipt gene is maternally expressed and continues to be transcribed throughout development and into adulthood, in a wide variety of tissues. Using an anti-zebrafish PI synthase polyclonal antiserum, we localized the protein throughout the developing eye, including the photoreceptor layer and lens cortical secondary fiber cells. As expected, the polyclonal antiserum revealed that the

  18. Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye.

    PubMed

    Murphy, Taylor R; Vihtelic, Thomas S; Ile, Kristina E; Watson, Corey T; Willer, Gregory B; Gregg, Ronald G; Bankaitis, Vytas A; Hyde, David R

    2011-10-01

    The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the cdipt-encoded PI synthase protein phenocopied the cdipt(lop/lop) mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of in vitro transcribed, wild-type cdipt mRNA into 1-4 cell stage cdipt(lop/lop) embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a cdipt retroviral-insertion allele, cdipt(hi559), exhibited similar lens and retinal abnormalities and failed to complement the cdipt(lop) mutant phenotype. To determine the initial cellular defects associated with the cdipt mutant, we examined homozygous cdipt(hi559/hi559) mutants prior to gross lens opacification at 6 dpf. The cdipt(hi559/hi559) mutants first exhibited photoreceptor layer disruption and photoreceptor cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. RT-PCR revealed that the cdipt gene is maternally expressed and continues to be transcribed throughout development and into adulthood, in a wide variety of tissues. Using an anti-zebrafish PI synthase polyclonal antiserum, we localized the protein throughout the developing eye, including the photoreceptor layer and lens cortical secondary fiber cells. As expected, the polyclonal antiserum

  19. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  20. Bone marrow-derived lung epithelial cells.

    PubMed

    Krause, Diane S

    2008-08-15

    Bone marrow-derived cells can take on the phenotype of epithelial cells and express epithelial-specific genes in multiple organs. Here, we focus on recent data on the appearance of marrow-derived epithelial cells in the adult lung. These findings have garnered significant skepticism because in most cases marrow-derived epithelial cells are very rare, the marrow cell of origin is not known, the techniques for detection have needed improvement, and there seem to be multiple mechanisms by which this occurs. Recent studies have focused on these concerns. Once these important concerns are addressed, further studies on the function(s) of these cells will need to be performed to determine whether this engraftment has any clinical significance-either beneficial or detrimental.

  1. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    Epithelial Stem Cells PRINCIPAL INVESTIGATOR: Peter D. Eirew CONTRACTING ORGANIZATION: British Columbia Cancer Agency...NUMBER Characterization of Human Mammary Epithelial Stem Cells 5b. GRANT NUMBER W81XWH-06-1-0702 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Abstract The mammary epithelium in normal adult female mice contains undifferentiated stem cells with extensive in vivo regenerative and self-renewal

  2. Functions of crystallins in and out of lens: Roles in elongated and post-mitotic cells

    PubMed Central

    Slingsby, Christine; Wistow, Graeme J.

    2014-01-01

    The vertebrate lens evolved to collect light and focus it onto the retina. In development, the lens grows through massive elongation of epithelial cells possibly recapitulating the evolutionary origins of the lens. The refractive index of the lens is largely dependent on high concentrations of soluble proteins called crystallins. All vertebrate lenses share a common set of crystallins from two superfamilies (although other lineage specific crystallins exist). The α-crystallins are small heat shock proteins while the β- and γ-crystallins belong to a superfamily that contains structural proteins of uncertain function. The crystallins are expressed at very high levels in lens but are also found at lower levels in other cells, particularly in retina and brain. All these proteins have plausible connections to maintenance of cytoplasmic order and chaperoning of the complex molecular machines involved in the architecture and function of cells, particularly elongated and post-mitotic cells. They may represent a suite of proteins that help maintain homeostasis in such cells that are at risk from stress or from the accumulated insults of aging. PMID:24582830

  3. Myb permits multilineage airway epithelial cell differentiation

    PubMed Central

    Pan, Jie-hong; Adair-Kirk, Tracy L.; Patel, Anand C.; Huang, Tao; Yozamp, Nicholas S.; Xu, Jian; Reddy, E. Premkumar; Byers, Derek E.; Pierce, Richard A.; Holtzman, Michael J.; Brody, Steven L.

    2014-01-01

    The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63+ basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps is poorly defined. Here we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb+ cells were identified as p63− and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63− population with failed maturation of Foxj1+ ciliated cells, as well as Scbg1a1+ and Muc5ac+ secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb+ cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63− Myb+ population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease. PMID:25103188

  4. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly

    PubMed Central

    Chaffee, Blake R.; Shang, Fu; Chang, Min-Lee; Clement, Tracy M.; Eddy, Edward M.; Wagner, Brad D.; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L.; Taylor, Allen

    2014-01-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation. PMID:25139855

  5. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

    PubMed

    Chaffee, Blake R; Shang, Fu; Chang, Min-Lee; Clement, Tracy M; Eddy, Edward M; Wagner, Brad D; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L; Taylor, Allen

    2014-09-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation. © 2014. Published by The Company of Biologists Ltd.

  6. The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation.

    PubMed

    Weber, Gregory F; Menko, A Sue

    2005-06-10

    The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.

  7. Purification of kidney epithelial cell growth inhibitors.

    PubMed Central

    Holley, R W; Böhlen, P; Fava, R; Baldwin, J H; Kleeman, G; Armour, R

    1980-01-01

    Two high molecular weight growth inhibitors have been isolated from the culture medium of BSC-1 cells, epithelial cells of African green monkey kidney. The purified kidney epithelial cell growth inhibitors, at ng/ml concentrations, reversibly arrest the growth of BSC-1 cells in the G1 phase of the cell cycle. Their action is selective; they are most active on BSC-1 cells, are less active as inhibitors of the growth of rat lung and human breast epithelial cells, and do not inhibit the growth of 3T3 mouse embryo fibroblasts ad human skin fibroblasts in culture. Their growth inhibitory action on BSC-1 cell cultures is counteracted by epidermal growth factor or calf serum. PMID:6969400

  8. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-08

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes.

  9. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed.

  10. Evidence for epithelial-mesenchymal transitions in adult liver cells.

    PubMed

    Sicklick, Jason K; Choi, Steve S; Bustamante, Marcia; McCall, Shannon J; Pérez, Elizabeth Hernández; Huang, Jiawen; Li, Yin-Xiong; Rojkind, Marcos; Diehl, Anna Mae

    2006-10-01

    Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.

  11. A stochastic model of eye lens growth.

    PubMed

    Šikić, Hrvoje; Shi, Yanrong; Lubura, Snježana; Bassnett, Steven

    2015-07-07

    The size and shape of the ocular lens must be controlled with precision if light is to be focused sharply on the retina. The lifelong growth of the lens depends on the production of cells in the anterior epithelium. At the lens equator, epithelial cells differentiate into fiber cells, which are added to the surface of the existing fiber cell mass, increasing its volume and area. We developed a stochastic model relating the rates of cell proliferation and death in various regions of the lens epithelium to deposition of fiber cells and radial lens growth. Epithelial population dynamics were modeled as a branching process with emigration and immigration between proliferative zones. Numerical simulations were in agreement with empirical measurements and demonstrated that, operating within the strict confines of lens geometry, a stochastic growth engine can produce the smooth and precise growth necessary for lens function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. A Stochastic Model of Eye Lens Growth

    PubMed Central

    Šikić, Hrvoje; Shi, Yanrong; Lubura, Snježana; Bassnett, Steven

    2015-01-01

    The size and shape of the ocular lens must be controlled with precision if light is to be focused sharply on the retina. The lifelong growth of the lens depends on the production of cells in the anterior epithelium. At the lens equator, epithelial cells differentiate into fiber cells, which are added to the surface of the existing fiber cell mass, increasing its volume and area. We developed a stochastic model relating the rates of cell proliferation and death in various regions of the lens epithelium to deposition of fiber cells and lens growth. Epithelial population dynamics were modeled as a branching process with emigration and immigration between various proliferative zones. Numerical simulations were in agreement with empirical measurements and demonstrated that, operating within the strict confines of lens geometry, a stochastic growth engine can produce the smooth and precise growth necessary for lens function. PMID:25816743

  13. Epithelial stem cells and intestinal cancer.

    PubMed

    Tan, Shawna; Barker, Nick

    2015-06-01

    The mammalian intestine is comprised of an epithelial layer that serves multiple functions in order to maintain digestive activity as well as intestinal homeostasis. This epithelial layer contains highly proliferative stem cells which facilitate its characteristic rapid regeneration. How these stem cells contribute to tissue repair and normal homeostasis are actively studied, and while we have a greater understanding of the molecular mechanisms and cellular locations that underlie stem cell regulation in this tissue, much still remains undiscovered. This review describes epithelial stem cells in both intestinal and non-intestinal tissues, as well as the strategies that have been used to further characterize the cells. Through a discussion of the current understanding of intestinal self-renewal and tissue regeneration in response to injury, we focus on how dysregulation of critical signaling pathways results in potentially oncogenic aberrations, and highlight issues that should be addressed in order for effective intestinal cancer therapies to be devised.

  14. Epithelial cell extrusion: Pathways and pathologies.

    PubMed

    Gudipaty, Swapna Aravind; Rosenblatt, Jody

    2016-05-19

    To remove dying or unwanted cells from an epithelium while preserving the barrier function of the layer, epithelia use a unique process called cell extrusion. To extrude, the cell fated to die emits the lipid Sphingosine 1 Phosphate (S1P), which binds the G-protein-coupled receptor Sphingosine 1 Phosphate receptor 2 (S1P2) in the neighboring cells that activates Rho-mediated contraction of an actomyosin ring circumferentially and basally. This contraction acts to squeeze the cell out apically while drawing together neighboring cells and preventing any gaps to the epithelial barrier. Epithelia can extrude out cells targeted to die by apoptotic stimuli to repair the barrier in the face of death or extrude live cells to promote cell death when epithelial cells become too crowded. Indeed, because epithelial cells naturally turn over by cell death and division at some of the highest rates in the body, epithelia depend on crowding-induced live cell extrusion to preserve constant cell numbers. If extrusion is defective, epithelial cells rapidly lose contact inhibition and form masses. Additionally, because epithelia act as the first line of defense in innate immunity, preservation of this barrier is critical for preventing pathogens from invading the body. Given its role in controlling constant cell numbers and maintaining barrier function, a number of different pathologies can result when extrusion is disrupted. Here, we review mechanisms and signaling pathways that control epithelial extrusion and discuss how defects in these mechanisms can lead to multiple diseases. We also discuss tactics pathogens have devised to hijack the extrusion process to infect and colonize epithelia.

  15. Epithelial monolayer culture system for real‐time single‐cell analyses

    PubMed Central

    Seo, Jong Bae; Moody, Mark; Koh, Duk‐Su

    2014-01-01

    Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single‐cell and subcellular levels, and can be extended to other cell types with minor modifications. PMID:24771696

  16. Epithelial monolayer culture system for real-time single-cell analyses.

    PubMed

    Seo, Jong Bae; Moody, Mark; Koh, Duk-Su

    2014-01-01

    Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single-cell and subcellular levels, and can be extended to other cell types with minor modifications.

  17. Deletion of DDB1 in mouse brain and lens leads to p53-dependent elimination of proliferating cells.

    PubMed

    Cang, Yong; Zhang, Jianxuan; Nicholas, Sally A; Bastien, Jayson; Li, Baojie; Zhou, Pengbo; Goff, Stephen P

    2006-12-01

    DDB1, a component of the Cul4 ubiquitin ligase complex, promotes protein ubiquitination in diverse cellular functions, including nuclear excision repair, regulation of the cell cycle, and DNA replication. To investigate its physiological significance, we generated mice with null and floxed alleles of the DDB1 gene. Here we report that null mutation of DDB1 caused early embryonic lethality, while conditional inactivation of the gene in brain and lens led to neuronal and lens degeneration, brain hemorrhages, and neonatal death. These defects stemmed from a selective elimination of nearly all proliferating neuronal progenitor cells and lens epithelial cells by apoptosis. The cell death was preceded by aberrant accumulation of cell cycle regulators and increased genomic instability and could be partially rescued by removal of the tumor suppressor protein p53. Our results indicate that DDB1 plays an essential role in maintaining viability and genomic integrity of dividing cells.

  18. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    PubMed

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  19. Epithelial: lamina propria lymphocyte interactions promote epithelial cell differentiation

    PubMed Central

    Dahan, Stephanie; Roda, Giulia; Pinn, David; Roth-Walter, Franziska; Kamalu, Okebugwu; Martin, Andrea P.; Mayer, Lloyd

    2010-01-01

    Background & Aims Lymphoepithelial interactions in the gut can occur in the epithelium and the sub-epithelial space. We asked whether Normal, Crohn’s Disease (CD) or Ulcerative colitis (UC) lamina propria lymphocytes (LPL) could promote intestinal epithelial cell (IEC) growth and differentiation. Methods T84 cells were co-cultured with freshly isolated LPL for varying periods. After removal of LPL, IECs were lysed and subjected to i) measurement of intestinal alkaline phosphatase (IAP) activity; ii) Western blot analysis for MAPK and Akt activation; and iii) Real Time-PCR to assess CDX2 mRNA levels. Tissue sections were immunostained for evidence of MAPK and PI3K activation, CDX2 and IAP; and CDX2 mRNA expression was assessed on human colonic biopsies. Results IAP activity was increased in T84 cells co-cultured for 8 days with Normal LPL (p<0.05), and even greater with CD LPL (p<0.001). Crypt IECs in active CD mucosa expressed IAP ex vivo. Phospho-MAPK (ERK1/2, p38, and JNK) and phospho-Akt were seen as early as 30 min after co-culture. MAPK activation was greatest in T84 cells co-cultured with CD LPL. There was a specific increase in P-p38 MAPK and P-Akt staining in the nuclei of crypt IECs in active vs inactive CD, normal mucosa and UC mucosa. CDX2 mRNA expression was increased in CD LPL co-cultured T84 cells which not correlated with the CDX2 protein localization ex vivo. Conclusion Our observations indicate that there is crosstalk between LPL and IECs, which leads to IEC differentiation. Moreover, in CD mucosa, the differentiation of IEC is accelerated. PMID:18045591

  20. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  1. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  2. Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

    PubMed

    Maddala, Rupalatha; Chauhan, Bharesh K; Walker, Christopher; Zheng, Yi; Robinson, Michael L; Lang, Richard A; Rao, Ponugoti V

    2011-12-01

    Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.

  3. Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

    PubMed Central

    Maddala, Rupalatha; Chauhan, Bharesh K.; Walker, Christopher; Zheng, Yi; Robinson, Michael L.; Lang, Richard A.; Rao, Ponugoti V.

    2011-01-01

    Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover. PMID:21945075

  4. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  5. Planar cell polarity in the mammalian eye lens

    PubMed Central

    Sugiyama, Yuki; Lovicu, Frank J

    2011-01-01

    The major role of the eye lens is to transmit and focus images onto the retina. For this function, the lens needs to develop and maintain the correct shape, notably, the precise curvature and high-level order and organization of its elements. The lens is mainly comprised of highly elongated fiber cells with hexagonal cross-sectional profiles that facilitate regular packing. Collectively, they form concentrically arranged layers around the anterior-posterior polar axis, and their convex curvature contributes to the spheroidal shape of the lens. Although the lens has been a popular system for developmental studies, little is known about the mechanism(s) that underlies the development of its exquisite three-dimensional cellular architecture. In this review, we will describe our recent work, which shows how planar cell polarity (PCP) operates in lens and contributes to its morphogenesis. We believe that the lens will be a useful model system to study PCP in general and gain insights into mechanisms that generate high-level cellular order during development. PMID:22027540

  6. Planar cell polarity in the mammalian eye lens.

    PubMed

    Sugiyama, Yuki; Lovicu, Frank J; McAvoy, John W

    2011-01-01

    The major role of the eye lens is to transmit and focus images onto the retina. For this function, the lens needs to develop and maintain the correct shape, notably, the precise curvature and high-level order and organization of its elements. The lens is mainly comprised of highly elongated fiber cells with hexagonal cross-sectional profiles that facilitate regular packing. Collectively, they form concentrically arranged layers around the anterior-posterior polar axis, and their convex curvature contributes to the spheroidal shape of the lens. Although the lens has been a popular system for developmental studies, little is known about the mechanism(s) that underlies the development of its exquisite three-dimensional cellular architecture. In this review, we will describe our recent work, which shows how planar cell polarity (PCP) operates in lens and contributes to its morphogenesis. We believe that the lens will be a useful model system to study PCP in general and gain insights into mechanisms that generate high-level cellular order during development.

  7. Cell fusion between gastric epithelial cells and mesenchymal stem cells results in epithelial-to-mesenchymal transition and malignant transformation.

    PubMed

    He, Xianghui; Li, Baosong; Shao, Yang; Zhao, Na; Hsu, Yiling; Zhang, Zhixiang; Zhu, Liwei

    2015-01-30

    The discovery of cancer stem cells and tumor heterogeneity prompted the exploration of additional mechanisms aside from genetic mutations for carcinogenesis and cancer progression. The aim of the present study was to investigate the effect of cell fusion between mesenchymal stem cells and the gastric epithelial cells in tumorigenesis. Cell fusion between cord blood mesenchymal stem cells and human gastric epithelial cells was performed in vitro. Cell scratch and transwell assays were performed to determine migration and invasion abilities of the hybrids. The expressions of epithelial-mesenchymal transition-related proteins and genes were analyzed by immunocytochemistry and real time quantitative PCR. Tumorigenesis of the hybrids was evaluated through in vivo inoculation in nude mice. Hybrids expressed the phenotypes of both donor cells. Aneuploidy was observed in 84.1% of cells. The hybrids showed increased proliferation, migration and invasion abilities compared with the parental cells. In addition, the expression of N-cadherin and vimentin in the hybrids was significantly higher than that of the epithelial cells, and the mRNA expression of the epithelial-mesenchymal transition-related genes, Twist and Slug, in the hybrids was also increased compared with that of the parental epithelial cells. Furthermore, the hybrids formed masses of epithelial origin with glandular structures in BALB/c nude mice. These findings suggest that cell fusion between gastric epithelial cells and mesenchymal stem cells may result in epithelial to mesenchymal transition and malignant transformation.

  8. Association of mesenchymal cells and immunoglobulins with differentiating epithelial cells

    PubMed Central

    Bukovsky, Antonin; Caudle, Michael R; Keenan, Jeffrey A; Upadhyaya, Nirmala B; Van Meter, Stuart E; Wimalasena, Jay; Elder, Robert F

    2001-01-01

    Background Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells either associate with epithelium basement membrane [pericytes and perivascular monocyte-derived cells (MDC)] or reside within epithelium (MDC and T cells). Although intraepithelial mesenchymal cells were suggested to contribute to the epithelium physiology, their association with particular steps in differentiation of epithelial cells, interactions among themselves, and their fate remain unclear. We studied epitopes of mesenchymal cells and their products (immunoglobulins) in stratified epithelium of uterine ectocervix, which is one of the prototypes of complete cellular differentiation from stem into the aged cells. Results Perivascular CD14 primitive MDC associated with basal (stem) epithelial cells. Thy-1 pericytes of microvasculature secreted intercellular vesicles, which associated with Ki67 postmitotic epithelial cells expressing MHC class I. Intraepithelial T cells showed an association with veiled type MDC [dendritic cell (DC) precursors] among parabasal cells, and exhibited fragmentation after entering intermediate (mature) epithelial layers. Mature DC secreted CD68 and exhibited fragmentation after reaching mid intermediate layers. Binding of IgM was detected at the top of each layer: in the upper parabasal, upper intermediate, and most surface epithelial cells. IgG was confined to the entire superficial layer. Conclusions These data suggest that the phylogenetically and ontogenetically developed hierarchy of mesenchymal cells (MDC, pericytes, T cells) and immunoglobulins (IgM, IgG) accompanies differentiation of epithelial cells from immature into the mature and aged phenotype. Further studies of an involvement of mesenchymal cells in the regulation of tissue homeostasis may bring novel approaches to the prevention and therapy of tissue dysfunctions characterized by permanent tissue immaturity (muscular dystrophy

  9. Protons sensitize epithelial cells to mesenchymal transition.

    PubMed

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M; Pluth, Janice M; Cucinotta, Francis A

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.

  10. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  11. Evaluation of Corneal Epithelial Healing Under Contact Lens with Spectral-Domain Anterior Segment Optical Coherence Tomography (SD-OCT)

    PubMed Central

    Pang, Claudine E; M, Vanathi; Tan, Donald T.H; Mehta, Jodhbir S

    2011-01-01

    Purpose: To describe a novel technique of using Spectral-domain (SD) anterior segment optical coherence tomography (SD-OCT) in the evaluation of corneal epithelial healing under a therapeutic contact lens (TCL) after lamellar keratoplasty and Epi-LASIK procedures. Design: Prospective, non-comparative, observational case series. Methods: Ten eyes of eight patients undergoing lamellar corneal transplantation and Epi-LASIK procedures at the Singapore National Eye Centre were included in the study. Ultra-high resolution SD-OCT scans of the cornea with a TCL in-situ were performed sequentially on the first, third and fifth day after procedure, with the RTVue (Optovue, Inc, Fremont, CA, USA), and the image findings were correlated with the clinical picture. Complete epithelial healing was verified with removal of TCL and fluorescein staining. Results: 5 eyes underwent Descemet’s stripping automated endothelial keratoplasty (DSAEK), 1 eye underwent deep anterior lamellar keratoplasty (DALK) and 4 eyes underwent Epi-LASIK. All eyes had complete epithelial healing with TCL in-situ by the third post-operative day. SD-OCT images were able to demonstrate the epithelial layer distinctly under the TCL in all cases. Conclusions: SD-OCT is a valuable imaging tool for monitoring the progression of epithelial healing with TCL in situ in patients following corneal surgical procedures. PMID:21686324

  12. Airway epithelial cells: current concepts and challenges.

    PubMed

    Crystal, Ronald G; Randell, Scott H; Engelhardt, John F; Voynow, Judith; Sunday, Mary E

    2008-09-15

    The adult human bronchial tree is covered with a continuous layer of epithelial cells that play a critical role in maintaining the conduit for air, and which are central to the defenses of the lung against inhaled environmental concomitants. The epithelial sheet functions as an interdependent unit with the other lung components. Importantly, the structure and/or function of airway epithelium is deranged in major lung disorders, including chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Investigations regarding the airway epithelium have led to many advances over the past few decades, but new developments in genetics and stem cell/progenitor cell biology have opened the door to understanding how the airway epithelium is developed and maintained, and how it responds to environmental stress. This article provides an overview of the current state of knowledge regarding airway epithelial stem/progenitor cells, gene expression, cell-cell interactions, and less frequent cell types, and discusses the challenges for future areas of investigation regarding the airway epithelium in health and disease.

  13. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

    Romero-Steiner, S; Witek, T; Balish, E

    1990-01-01

    Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation. PMID:2298877

  14. CCN1 induces a reversible epithelial-mesenchymal transition in gastric epithelial cells.

    PubMed

    Chai, Jianyuan; Norng, Manith; Modak, Cristina; Reavis, Kevin M; Mouazzen, Wasim; Pham, Jennifer

    2010-08-01

    CCN1 is a matricellular protein that activates many genes related to wound healing and tissue remodeling in fibroblasts, but its effect on epithelial cells remains unclear. This study examined the role of CCN1 in epithelial wound healing using rat gastric epithelial cells and rat stomach ulcer as in vitro and in vivo models, respectively. We found that CCN1 expression is highly upregulated in the epithelial cells adjacent to a wound and remains high until the wound is healed. Upregulation of CCN1 activates a transient epithelial-mesenchymal transition in the epithelial cells at the migrating front and drives wound closure. Once the wound is healed, these epithelial cells and their progeny can resume their original epithelial phenotype. We also found that CCN1-induced E-cadherin loss is not due to transcriptional regulation but rather protein degradation due to the collapse of adherens junctions, which is contributed by beta-catenin translocation. CCN1-activated integrin-linked kinase mediates this process. Finally, our in vivo study showed that locally neutralizing CCN1 drastically impairs wound closure, whereas local injection of recombinant CCN1 protein induces expression of vimentin and smooth muscle alpha-actin in normal gastric mucosal epithelial cells and accelerates re-epithelialization during ulcer healing. In conclusion, our study indicates that CCN1 can induce reversible epithelial-mesenchymal transition, and this feature may have great value for clinical wound healing.

  15. Isolation of epithelial cells with hepatobiliary phenotype.

    PubMed

    Castorina, Sergio; Luca, Tonia; Torrisi, Antonella; Privitera, Giovanna; Panebianco, Mariangela

    2008-01-01

    The regenerative capacity of the liver after partial hepatectomy or chemical injury is well known. In human liver, the resident progenitor cells are called "hepatic progenitor cells" (HPCs) while the term "oval cells" should be discouraged in order to indicate the stem cell compartment. The aim of our study was first to analyse the cellular aspects of liver regeneration through differentiation in cholangiocytes and hepatocytes, and then to characterise resident progenitor cells, using "primary cultured hepatocytes" derived from healthy adult human livers. Human hepatocytes were isolated from fresh surgical specimens of patients who underwent hepatic resections in our Clinical Centre surgery operating room. Hepatic differentiation and function were analysed by immunocytochemistry techniques and the presence of liver epithelial cell populations within normal adult human liver, was demonstrated by immunohistochemistry analysis. These cells expanded in vitro and showed the capacity for self-renewal and multipotent differentiation. Human liver stem cells expressed several mesenchymal markers, such as CD44, but not haematopoietic stem cell markers. In addition, these cells expressed alpha-fetoprotein, albumin, CK7 and CK19, indicating a partial commitment to hepatic and biliary cells. Interestingly the expression of both hepatocytes and biliary markers in HPCs reflects the bipotential nature of the hepatic stem cells toward both the hepatic and biliary lineage. According to their immature and bipotential phenotype, hepatic epithelial cells might represent a pool of precursors in the healthy human adult liver.

  16. The human airway epithelial basal cell transcriptome.

    PubMed

    Hackett, Neil R; Shaykhiev, Renat; Walters, Matthew S; Wang, Rui; Zwick, Rachel K; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G

    2011-05-04

    The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.

  17. The Human Airway Epithelial Basal Cell Transcriptome

    PubMed Central

    Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

    2011-01-01

    Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

  18. Reversible transdifferentiation of alveolar epithelial cells.

    PubMed

    Danto, S I; Shannon, J M; Borok, Z; Zabski, S M; Crandall, E D

    1995-05-01

    Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.

  19. Disassembly of the lens fiber cell nucleus to create a clear lens: The p27 descent.

    PubMed

    Rowan, Sheldon; Chang, Min-Lee; Reznikov, Natalie; Taylor, Allen

    2017-03-01

    The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFCD). In contrast to the disassembly and reassembly of the cell nucleus during mitosis, LFCD is a unidirectional process that culminates in destruction of the fiber cell nucleus. Whereas p27(Kip1), the cyclin-dependent kinase inhibitor, is upregulated during formation of LFC in the outermost cortex, in the inner cortex, in the nascent organelle free zone, p27(Kip1) is degraded, markedly activating cyclin-dependent kinase 1 (Cdk1). This process results in phosphorylation of nuclear Lamins, dissociation of the nuclear membrane, and entry of lysosomes that liberate DNaseIIβ (DLAD) to cleave chromatin. Multiple cellular pathways, including the ubiquitin proteasome system and the unfolded protein response, converge on post-translational regulation of p27(Kip1). Mutations that impair these pathways are associated with congenital cataracts and loss of LFCD. These findings highlight new regulatory nodes in the lens and suggest that we are close to understanding this fascinating terminal differentiation process. Such knowledge may offer a new means to confront proliferative diseases including cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  1. Particle irradiation induces FGF2 expression in normal human lens cells

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Bjornstad K, A.; Chang, E.; McNamara, M.; Barcellos-Hoff, M. H.; Lin, S. P.; Aragon, G.; Polansky, J. R.; Lui, G. M.; Blakely, E. A.

    2000-01-01

    Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

  2. Particle irradiation induces FGF2 expression in normal human lens cells

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Bjornstad K, A.; Chang, E.; McNamara, M.; Barcellos-Hoff, M. H.; Lin, S. P.; Aragon, G.; Polansky, J. R.; Lui, G. M.; Blakely, E. A.

    2000-01-01

    Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

  3. Force mapping in epithelial cell migration

    PubMed Central

    du Roure, Olivia; Saez, Alexandre; Buguin, Axel; Austin, Robert H.; Chavrier, Philippe; Siberzan, Pascal; Ladoux, Benoit

    2005-01-01

    We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multiple-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 μm) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior. PMID:15695588

  4. Isolation by Size of Epithelial Tumor Cells

    PubMed Central

    Vona, Giovanna; Sabile, Abdelmajid; Louha, Malek; Sitruk, Veronique; Romana, Serge; Schütze, Karin; Capron, Frédérique; Franco, Dominique; Pazzagli, Mario; Vekemans, Michel; Lacour, Bernard; Bréchot, Christian; Paterlini-Bréchot, Patrizia

    2000-01-01

    We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine. PMID:10623654

  5. Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors.

    PubMed

    Lorber, Barbara; Berry, Martin; Logan, Ann

    2005-04-01

    In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.

  6. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  7. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  8. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    PubMed Central

    Simon, R H; DeHart, P D; Todd, R F

    1986-01-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing. Images PMID:3771800

  9. Traction forces exerted by epithelial cell sheets

    NASA Astrophysics Data System (ADS)

    Saez, A.; Anon, E.; Ghibaudo, M.; du Roure, O.; Di Meglio, J.-M.; Hersen, P.; Silberzan, P.; Buguin, A.; Ladoux, B.

    2010-05-01

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  10. Traction forces exerted by epithelial cell sheets.

    PubMed

    Saez, A; Anon, E; Ghibaudo, M; du Roure, O; Di Meglio, J-M; Hersen, P; Silberzan, P; Buguin, A; Ladoux, B

    2010-05-19

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  11. Cells of Origin of Epithelial Ovarian Cancers

    DTIC Science & Technology

    2015-09-01

    lethal malignancy of the female reproductive system , largely due to the fact that most EOCs are diagnosed only after the cancer has metastasized into the...Epithelial ovarian cancer (EOC) is the most lethal malignancy of the female reproductive system , largely due to the fact that most EOCs are diagnosed only...ovarian cancer by defined multiple genetic changes in a mouse model system . Cancer Cell 1, 53-62. Quartuccio, S.M., Lantvit, D.D., Bosland, M.C., and

  12. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  13. Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells

    PubMed Central

    Maloum, Faïza; Allaire, Joannie M.; Gagné-Sansfaçon, Jessica; Roy, Evelyne; Belleville, Karine; Sarret, Philippe; Morisset, Jean; Carrier, Julie C.; Mishina, Yuji; Kaestner, Klaus H.

    2011-01-01

    Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1aΔGEC mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1aΔGEC mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia. PMID:21415412

  14. Potential pre-cataractous markers induced by low-dose radiation effects in cultured human lens cells

    NASA Astrophysics Data System (ADS)

    Blakely, E.; McNamara, M.; Bjornstad, K.; Chang, P.

    The human lens is one of the most radiosensitive organs of the body. Cataract, the opacification of the lens, is a late-appearing response to radiation damage. Recent evidence indicates that exposure to relatively low doses of space radiation are associated with an increased incidence and early appearance of human cataracts (Cucinotta et al., Radiat. Res. 156:460-466, 2001). Basic research in this area is needed to integrate the early responses of various late-responding tissues into our understanding and estimation of radiation risk for space travel. In addition, these studies may contribute to the development of countermeasures for the early lenticular changes, in order to prevent the late sequelae. Radiation damage to the lens is not life threatening but, if severe, can affect vision unless surgically corrected with synthetic lens replacement. The lens, however, may be a sensitive detector of radiation effects for other cells of ectodermal origin in the body for which there are not currently clear endpoints of low-dose radiation effects. We have investigated the dose-dependent expression of several radiation-responsive endpoints using our in vitro model of differentiating human lens epithelial cells (Blakely et al., Investigative Ophthalmology &Visual Sciences, 41(12):3898-3907, 2000). We have investigated radiation effects on several gene families that include, or relate to, DNA damage, cytokines, cell-cycle regulators, cell adhesion molecules, cell cytoskeletal function and apoptotic cell death. In this paper we will summarize some of our dose-dependent data from several radiation types, and describe the model of molecular and cellular events that we believe may be associated with precataractous events in the human lens after radiation exposure. This work was supported by NASA Grant #T-965W.

  15. The Notch Signaling Pathway Controls the Size of the Ocular Lens by Directly Suppressing p57Kip2 Expression▿ †

    PubMed Central

    Jia, Junling; Lin, Min; Zhang, Lingna; York, J. Philippe; Zhang, Pumin

    2007-01-01

    The size of an organ must be tightly controlled so that it fits within an organism. The mammalian lens is a relatively simple organ composed of terminally differentiated, amitotic lens fiber cells capped on the anterior surface by a layer of immature, mitotic epithelial cells. The proliferation of lens epithelial cells fuels the growth of the lens, thus controling the size of the lens. We report that the Notch signaling pathway defines the boundary between proliferation and differentiation in the developing lens. The loss of Notch signaling results in the loss of epithelial cells to differentiation and a much smaller lens. We found that the Notch effector Herp2 is expressed in lens epithelium and directly suppresses p57Kip2 expression, providing a molecular link between Notch signaling and the cell cycle control machinery during lens development. PMID:17709399

  16. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

  17. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  18. Detection of Bone Marrow Derived Lung Epithelial Cells

    PubMed Central

    Kassmer, Susannah H.; Krause, Diane S.

    2010-01-01

    Studies on the ability of bone marrow derived cells to adopt the morphology and protein expression of epithelial cells in vivo have expanded rapidly over the last decade, and hundreds of publications report that bone marrow derived cells can become epithelial cells of multiple organs including lung, liver, GI tract, skin, pancreas and others. In this review, we critically evaluate the literature related to engraftment of bone marrow derived cells as epithelial cells in the lung. Over 40 manuscripts focused on whether bone marrow cells can differentiate into lung epithelial cells have been published, nearly all of which claim to identify marrow derived epithelial cells. A few investigations have concluded that no such cells are present and that the phenomenon of marrow derived epithelial cells is based on detection artifacts. Here we discuss the problems that exist in published papers identifying marrow derived epithelial cells, and propose standards for detection methods that provide the most definitive data. Identification of BM derived epithelial cells requires reliable and sensitive techniques for their detection, which must include cell identification based on the presence of an epithelial marker and the absence of blood cell markers as well as a marker for donor BM origin. In order for these studies to be rigorous, they must also use approaches to rule out cell overlap by microscopy or single cell isolation. Once these stringent criteria for identification of marrow derived epithelial cells are used universally, then the field can move forward to address the critical questions regarding which bone marrow derived cells are responsible for engraftment as epithelial cells, the mechanisms by which this occurs, whether these cells play a role in normal tissue repair, and whether specific cell subsets can be used for therapeutic benefit. PMID:20447442

  19. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans.

  20. Expression and function of PDGF-α in columnar epithelial cells of age-related cataracts patients.

    PubMed

    Wei, J; Tang, H; Xu, Z Q; Li, B; Xie, L Q; Xu, G X

    2015-10-27

    We studied the expression and function of platelet-derived growth factor A (PDGF-α) in the lens epithelial cells of cataracts patients. Ninety age-related cataracts patients were recruited in our hospital between January 2012 and January 2014. The expression levels of platelet-derived growth factor receptor (PDGFR) in the anterior capsule of the lens at different degrees of turbidity, and PDGF-α in the aqueous humor were detected. A human lens epithelium cell line was also cultured and studied. To investigate its function, PDGF-α was used to treat a PDGFR-silenced human lens epithelium cell line to observe changes in the proliferation, transfer, and epithelial mesenchymal transition (EMT). The expression of PDGF-α and its receptor increased in patients with more serious cataracts. Lens epithelium cells stimulated by PDGF-α showed greater proliferation and migration. The degree of EMT was also upregulated in cells stimulated by PDGF-α. However, silencing the expression of PDGFR inhibited the effects. The development and severity of age-related cataracts was related to the secretion and expression of PDGF-α. This may be a new therapeutic target for cataracts treatment.

  1. Influence of a Therapeutic Soft Contact Lens on Epithelial Healing, Visual Recovery, Haze, and Pain After Photorefractive Keratectomy.

    PubMed

    Taneri, Suphi; Oehler, Saskia; MacRae, Scott; Dick, H Burkhard

    2016-08-30

    To examine the influence of a therapeutic soft contact lens (TSCL) after alcohol-assisted photorefractive keratectomy (PRK) on visual recovery, epithelial closure, pain perception, and haze formation. Prospective, randomized, single-center, contralateral eye, patient-masked study. Same surgeon treated 15 patients with bilateral PRK using a Vidaurri Fluid Retention Ring (Katena) with 8.7 mm inner diameter to apply ethanol 18% for 30 sec. Each patient received a TSCL (Balafilcon A, PureVision, Bausch & Lomb) in a randomized fashion in one eye only. Without knowing which eye had a contact lens, four patients preferred the eye with TSCL so much that these patients wanted a TSCL inserted in the second eye as well. Uncorrected distance visual acuity improved postoperatively from day 1 to 3 months from 0.61 to 1.18 with TSCL and from 0.21 to 1.04 without TSCL. Uncorrected distance visual acuity was statistically significantly better on days 1 and 2 with TSCL. Epithelial defect was on average smaller with TSCL. Epithelium was closed at day 4 in all eyes except one without TSCL. Pain perception was significantly lower with TSCL on days 1 and 2. Haze levels after 3 months were significantly lower with TSCL (with 0.09/without 0.45, respectively). One eye without TSCL had haze grade 2 (Fantes). A TSCL had statistically significantly and clinically meaningful beneficial effects in the first days in visual recovery and pain perception and after 3 months in haze formation. Epithelial healing was slightly quicker with the use of TSCLs (not statistically, but clinically significantly).

  2. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

    PubMed Central

    Vielfort, Katarina; Weyler, Linda; Söderholm, Niklas; Engelbrecht, Mattias; Löfmark, Sonja; Aro, Helena

    2013-01-01

    We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells. PMID:23675492

  3. Contact lens biofuel cell tested in a synthetic tear solution.

    PubMed

    Reid, Russell C; Minteer, Shelley D; Gale, Bruce K

    2015-06-15

    A contact lens biofuel cell was fabricated using buckypaper electrodes cured on a silicone elastomer soft contact lens. The buckypaper anode consisted of poly(methylene green) and a hydrogel matrix containing lactate dehydrogenase and nicotinamide adenine dinucleotide hydrate (NAD(+)). The buckypaper cathode was modified with 1-pyrenemethyl anthracene-2-carboxylate, and then bilirubin oxidase was immobilized within a polymer. Contact lens biofuel cell testing was performed in a synthetic tear solution at 35°C. The open circuit voltage was 0.413±0.06 V and the maximum current and power density were 61.3±2.9 µA cm(-2) and 8.01±1.4 µWc m(-2), respectively. Continuous operation for 17h revealed anode instability as output current rapidly decreased in the first 4h and then stabilized for the next 13 h. The contact lens biofuel cell presented here is a step toward achieving self-powered electronic contact lenses and ocular devices with an integrated power source.

  4. Cell reintegration: Stray epithelial cells make their way home.

    PubMed

    Wilson, Tyler J; Bergstralh, Dan T

    2017-06-01

    Ongoing work shows that misplaced epithelial cells have the capacity to reintegrate back into tissue layers. This movement appears to underlie tissue stability and may also control aspects of tissue structure. A recent study reveals that cell reintegration in at least one tissue, the Drosophila follicular epithelium, is based on adhesion molecules that line lateral cell surfaces. In this article we will review these observations, discuss their implications for epithelial tissue development and maintenance, and identify future directions for study. © 2017 WILEY Periodicals, Inc.

  5. Phenotypic plasticity in normal breast derived epithelial cells

    PubMed Central

    2014-01-01

    Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools. PMID:24915897

  6. Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

    PubMed Central

    Hu, Fanlei; Zhang, Li; Zheng, Jie; Zhao, Ling; Huang, Jing; Shao, Wenwei; Liao, Qinyuan; Ma, Teng; Geng, Li; Yin, C. Cameron; Qiu, Xiaoyan

    2012-01-01

    It is well known that B-1 B cells are the main cell type that is responsible for the production of natural immunoglobulin M (IgM) and can respond to infection by increasing IgM secretion. However, we unexpectedly found that some epithelial cells also can express rearranged IgM transcript that has natural IgM characteristics, such as germline-encoded and restricted rearrangement patterns. Here we studied IgM expression in human non-B cells and found that IgM was frequently expressed by many human epithelial cancer cells as well as non-cancer epithelial cells. Moreover, CD79A and CD79B, two molecules that are physically linked to membranous IgM on the surface of B cells to form the B cell antigen receptor complex, were also expressed on the cell surface of epithelial cancer cells and co-located with IgM. Like the natural IgM, the epithelial cancer cell-derived IgM recognized a series of microbial antigens, such as single-stranded DNA, double-stranded DNA, lipopolysaccharide, and the HEp-2 cell antigen. More important, stimulation of the toll-like receptor 9 (TLR9), which mimics bacterial infection, substantially increased the secretion of IgM in human epithelial cancer cells. These findings indicate that human epithelial cancer cells as well as non-cancer epithelial cells can spontaneously produce IgM with natural antibody activity. PMID:23251529

  7. Promoter methylation in epithelial-enriched and epithelial-depleted cell populations isolated from breast milk.

    PubMed

    Browne, Eva P; Dinc, Signem E; Punska, Elizabeth C; Agus, Sami; Vitrinel, Ayca; Erdag, Gulay Ciler; Anderton, Douglas L; Arcaro, Kathleen F; Yilmaz, Bayram

    2014-11-01

    Breast cancer is the most frequently diagnosed cancer among Turkish women and both the incidence and associated mortality appear to be increasing. Of particular concern is the percentage of young women diagnosed with breast cancer; roughly 20% of all breast cancer diagnoses in Turkey are in women younger than 40 years. Increased DNA methylation in the promoter region of tumor suppressor genes is a promising molecular biomarker, and human milk provides exfoliated breast epithelial cells appropriate for DNA methylation analyses. Comparisons between DNA methylation patterns in epithelial (epithelial-enriched) and nonepithelial (epithelial-depleted) cell fractions from breast milk have not been reported previously. In the present study, we examined promoter methylation of 3 tumor suppressor genes in epithelial-enriched and epithelial-depleted cell fractions isolated from breast milk of 43 Turkish women. Percentage methylation in the promoter region of Rass association domain family 1 (RASSF1), secreted frizzle related protein 1 (SFRP1), and glutathione-S-transferase class pi 1 was determined by pyrosequencing of the epithelial-enriched and epithelial-depleted cell fractions. Pyrosequencing identified a few subjects with significantly increased methylation in 1 or more genes. There was little correlation between the 2 cell fractions within individuals; only 1 woman had increased methylation for 1 gene (SFRP1) in both her enriched and depleted cell fractions. Methylation was positively associated with age for SFRP1 (epithelial-depleted fraction) and with body mass index for RASSF1 (epithelial-enriched cell fraction), respectively. Overall, results show that the methylation signals vary between different cell types in breast milk and suggest that breast milk can be used to assess DNA methylation patterns associated with increased breast cancer risk. © The Author(s) 2014.

  8. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

    Appendix……………………………………………………………………………… 11 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A method for... Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability...Abstracts Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turshvili, Sam Aparicio , Joanne Emerman and Connie Eaves, “Identification of Human Mammary

  9. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  10. Transgenic Expression of AQP1 in the Fiber Cells of AQP0 Knockout Mouse: Effects on Lens Transparency

    PubMed Central

    Varadaraj, K.; Kumari, S.S.; Mathias, R.T.

    2010-01-01

    Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0-/-) mouse? To investigate, we generated a transgenic wild type mouse line expressing AQP1 in the fiber cells using αA-crystallin promoter. These transgenic mice (TgAQP1+/+) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1+/+/AQP0-/-) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal

  11. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

  12. [Research progress of corneal epithelial basal cells and basement membrane].

    PubMed

    Qu, J H; Sun, X G

    2016-09-11

    The cylinder cells at the bottom of corneal epithelial cells are basal cells. Their cytoplasm contains keratin intermediate filament which is important in secretion of basement membrane. Corneal epithelial dysfunction due to diabetes or ocular surgery is intimately related with basal cell abnormality. Corneal epithelial basement membrane is a highly specific extracellular matrix which is made up of lamina lucida and lamina densa. It plays an extremely important role in renewal and restoration. Many ocular abnormalities and diseases have been described to relate to the corneal epithelial basement membrane, such as traumatic recurrent corneal erosion, corneal dystrophy and keratoconus. (Chin J Ophthalmol, 2016, 52: 703-707).

  13. Disruption of Rest Leads to the Early Onset of Cataracts with the Aberrant Terminal Differentiation of Lens Fiber Cells

    PubMed Central

    Aoki, Hitomi; Ogino, Hajime; Tomita, Hiroyuki; Hara, Akira; Kunisada, Takahiro

    2016-01-01

    REST (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells in vitro by preventing precocious expression of neuronal genes. REST expression was then decreased in developing neurons to down-regulate neuronal genes which allow their maturation. However, the function of REST during neurogenesis in vivo remains to be elucidated because of the early embryonic lethal phenotype of conventional Rest knockout mice. In order to investigate the role of REST in ocular tissues, we generated and examined the mice evoking genetic ablation to Rest specifically to neural tissues including ocular tissue. We used a Sox1-Cre allele to excise the floxed Rest gene in the early neural tissues including the lens and retinal primordia. The resulting Rest conditional knockout (CKO) and co cntrol mice were used in comparative morphological, histological, and gene expression analyses. Rest CKO mice had an abnormal lens morphology after birth. The proliferation of lens epithelial cells was likely to be slightly reduced, and vacuoles formed without a visible increase in apoptotic cells. Although the aberrant expression of late onset cataract marker proteins was not detected, the expression of Notch signaling-related genes including a previously identified REST-target gene was up-regulated around birth, and this was followed by the down-regulated expression of lens fiber regulators such as c-Maf and Prox1. Rest CKO induces a unique cataract phenotype just after birth. Augmented Notch signaling and the down-regulated expression of lens fiber regulator genes may be responsible for this phenotype. Our results highlight the significance of REST function in lens fiber formation, which is necessary for maintaining an intact lens structure. PMID:27631609

  14. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.

  15. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  16. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

    Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.

  17. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  18. Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

    PubMed Central

    Vanni, Cristina; Ognibene, Marzia; Finetti, Federica; Mancini, Patrizia; Cabodi, Sara; Segalerba, Daniela; Torrisi, Maria Rosaria; Donnini, Sandra; Bosco, Maria Carla; Varesio, Luigi; Eva, Alessandra

    2015-01-01

    The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression. PMID:25723869

  19. Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion.

    PubMed

    Vanni, Cristina; Ognibene, Marzia; Finetti, Federica; Mancini, Patrizia; Cabodi, Sara; Segalerba, Daniela; Torrisi, Maria Rosaria; Donnini, Sandra; Bosco, Maria Carla; Varesio, Luigi; Eva, Alessandra

    2015-01-01

    The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression.

  20. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  1. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  2. Epithelial Cell Innate Response to Candida albicans

    PubMed Central

    Naglik, J.R.; Moyes, D.

    2011-01-01

    With the advent of treatments and diseases such as AIDS resulting in increasing numbers of patients with suppressed immune systems, fungal diseases are an escalating problem. Candida albicans is the most common of these fungal pathogens, causing infections in many of these patients. It is therefore important to understand how immunity to this fungus is regulated and how it might be manipulated. Although work has been done to identify the receptors, fungal moieties, and responses involved in anti-Candida immunity, most studies have investigated interactions with myeloid or lymphoid cells. Given that the first site of contact of C. albicans with its host is the mucosal epithelial surface, recent studies have begun to focus on interactions of C. albicans with this site. The results are startling yet in retrospect obvious, indicating that epithelial cells play an important role in these interactions, initiating responses and even providing a level of protection. These findings have obvious implications, not just for fungal pathogens, but also for identifying how host organisms can distinguish between commensal and pathogenic microbes. This review highlights some of these recent findings and discusses their importance in the wider context of infection and immunity. PMID:21441481

  3. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions.

    PubMed

    Van Landeghem, Laurianne; Mahé, Maxime M; Teusan, Raluca; Léger, Jean; Guisle, Isabelle; Houlgatte, Rémi; Neunlist, Michel

    2009-11-02

    Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions.

  4. Dedifferentiation of committed epithelial cells into stem cells in vivo

    PubMed Central

    Tata, Purushothama Rao; Mou, Hongmei; Pardo-Saganta, Ana; Zhao, Rui; Prabhu, Mythili; Prabhu, Mythili; Law, Brandon M.; Vinarsky, Vladimir; Cho, Josalyn L.; Breton, Sylvie; Sahay, Amar; Medoff, Benjamin D.; Rajagopal, Jayaraj

    2014-01-01

    Summary Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes, and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. We now present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. Following the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts to repair epithelial injury. Indeed, single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. In contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate was inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may play a more general role in the regeneration of many tissues and in multiple disease states, notably cancer. PMID:24196716

  5. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  6. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  7. Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

    PubMed Central

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

    2017-01-01

    Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1

  8. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  9. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  10. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  11. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    PubMed Central

    Bauer, Rebecca N.; Müller, Loretta; Brighton, Luisa E.; Duncan, Kelly E.

    2015-01-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell–Mac coculture model to investigate how epithelial cell–derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell–Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell–derived signals are important determinants of Mac immunophenotype and response to O3. PMID:25054807

  12. Ouabain modulates epithelial cell tight junction

    PubMed Central

    Larre, Isabel; Lazaro, Amparo; Contreras, Ruben G.; Balda, Maria S.; Matter, Karl; Flores-Maldonado, Catalina; Ponce, Arturo; Flores-Benitez, David; Rincon-Heredia, Ruth; Padilla-Benavides, Teresita; Castillo, Aída; Shoshani, Liora; Cereijido, Marcelino

    2010-01-01

    Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell–cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K+ pumping nor disturb the K+ balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell–cell contacts. PMID:20534449

  13. Mechanical stretch triggers rapid epithelial cell division through Piezo1.

    PubMed

    Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

    2017-03-02

    Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

  14. Chromatin Degradation in Differentiating Fiber Cells of the Eye Lens

    PubMed Central

    Bassnett, Steven; Mataic, Danijela

    1997-01-01

    During development, the lens of the eye becomes transparent, in part because of the elimination of nuclei and other organelles from the central lens fiber cells by an apoptotic-like mechanism. Using confocal microscopy we showed that, at the border of the organelle-free zone (OFZ), fiber cell nuclei became suddenly irregular in shape, with marginalized chromatin. Subsequently, holes appeared in the nuclear envelope and underlying laminae, and the nuclei collapsed into condensed, spherical structures. Nuclear remnants, containing DNA, histones, lamin B2, and fragments of nuclear membrane, were detected deep in the OFZ. We used in situ electrophoresis to demonstrate that fragmented DNA was present only in cells bordering the OFZ. Confocal microscopy of terminal deoxynucleotidyl transferase (TdT)–labeled lens slices confirmed that DNA fragmentation was a relatively late event in fiber differentiation, occurring after the loss of the nuclear membrane. DNA fragments with 3′-OH or 3′-PO4 ends were not observed elsewhere in the lens under normal conditions, although they could be produced by pretreatment with DNase I or micrococcal nuclease, respectively. Dual labeling with TdT and an antibody against protein disulfide isomerase, an ER-resident protein, revealed a distinct spatial and temporal gap between the disappearance of ER and nuclear membranes and the onset of DNA degradation. Thus, fiber cell chromatin disassembly differs significantly from classical apoptosis, in both the sequence of events and the time course of the process. The fact that DNA degradation occurs only after the disappearance of mitochondrial, ER, and nuclear membranes suggests that damage to intracellular membranes may be an initiating event in nuclear breakdown. PMID:9105035

  15. Elemental profiles in Emory mouse lens

    SciTech Connect

    Bagchi, M.; Emanuel, K. )

    1991-01-01

    Energy dispersive x-ray microprobe analysis was used to determine the distribution of chloride, potassium, phosphorus and sulfur in the epithelial cells of the lenses obtained from 3 to 7 month old Emory mice and 7 month old cataract resistant strain of Emory mice. Rapidly frozen lenses were fractured in the frozen state and lyophilized. The anterior epithelial cells were analyzed from equator to equator. The results show that the epithelial cells of the 7 month old Emory mouse lens have considerably higher amounts of chloride, sulfur, potassium and phosphorus. Presence of increased amount of potassium in the epithelial cells is intriguing. The data obtained from these experiments show that the changes in the elemental levels of epithelial cells are similar to observed alteration found in the lens fiber mass of 7 month old Emory mouse.

  16. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.

    PubMed

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-07-19

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity.

  17. Unique and analogous functions of aquaporin O for fiber cell architecture and ocular lens transparency

    SciTech Connect

    Kumari, S.S.; Eswaramoorthy, S.; Mathias, R. T.; Varadaraj, K.

    2011-09-01

    Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein. To validate this notion, we determined if wild type (WT) lens ultrastructure and fibercell adhesion are different in AQP0{sup -/-}, and TgAQP1{sup +/+}/AQP0{sup -/-} mice that transgenically express AQP1 (TgAQP1) in fibercells without AQP0 (AQP0{sup -/-}). In WT, lenses were transparent with 'Y' sutures. Fibers contained opposite end curvature, lateral interdigitations, hexagonal shape, and were arranged as concentric growth shells. AQP0{sup -/-}lenses were cataractous, lacked 'Y' sutures, ordered packing and well-defined lateral interdigitations. TgAQP1{sup +/+}/AQP0{sup -/-} lenses showed improvement in transparency and lateral interdigitations in the outer cortex while inner cortex and nuclear fibers were severely disintegrated. Transmission electron micrographs exhibited tightly packed fibercells in WT whereas AQP0{sup -/-} and TgAQP1{sup +/+}/AQP0{sup -/-}lenses had wide extracellular spaces. Fibers were easily separable by teasing in AQP0{sup -/-} and TgAQP1{sup +/+}/AQP0{sup -/-}lenses compared to WT. Our data suggest that the increased water permeability through AQP1 does not compensate for loss of AQP0 expression in TgAQP1{sup +/+}/AQP0{sup -/-} mice. Fibercell AQP0 expression is required to maintain their organization, which is a requisite for lenstransparency. AQP0 appears necessary for cell-to-cell adhesion and thereby to minimize light scattering since in the AQP0{sup -/-} and TgAQP1{sup +/+}/AQP0{sup -/-} lenses, fiber cell disorganization was evident.

  18. Time-specific blockade of PDGFR with Imatinib (Glivec®) causes cataract and disruption of lens fiber cells in neonatal mice.

    PubMed

    Zhou, Yin-Pin; He, Yang-Tao; Chen, Cheng-Li; Ji, Jun; Niu, Jian-Qin; Wang, Han-Zhi; Li, Shi-Feng; Huang, Lan; Mei, Feng

    2011-03-01

    This study aimed at investigating the response of lens epithelial cells in postnatal mice to Imatinib (Glivec®, a potent inhibitor of platelet-derived growth factor receptor (PDGFR)) treatment. Mouse eyes were sampled 10 days after administration of Imatinib (0.5 mg·g(-1)·day(-1)) for 3 days, at either 7, 14, or 21 days postpartum. Structural changes of lens were revealed by routine H.E. staining. Levels of proliferation and apoptosis were revealed by BrdU incorporation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively, and immunofluorescent staining with anti-PDGFRα antibody was carried out on the sections of eyeball. PDGFRα and p-PDGFRαprotein levels were evaluated by Western blot. Our results indicated that administration of Imatinib led to blockade of PDGFR signaling. Formation of cataracts was found only in those mice where treatment started from 7 days postpartum (P7), but was not observed in those samples from P14 nor P21. Fiber cells were disorganized in cataract lens core as observed histologically, and migration of epithelial cells was also inhibited. No apoptosis was detected with the TUNEL method. Our results indicated blockade of PDGFR at the neonatal stage (P7) would lead to cataracts and lens fiber cells disorganization, suggesting that PDGFR signaling plays a time-specific and crucial role in the postnatal development of lens in the mouse, and also may provide a new approach to produce a congenital cataract animal model.

  19. Contact Lens-induced Limbal Stem Cell Deficiency.

    PubMed

    Rossen, Jennifer; Amram, Alec; Milani, Behrad; Park, Dongwook; Harthan, Jennifer; Joslin, Charlotte; McMahon, Timothy; Djalilian, Ali

    2016-10-01

    Limbal stem cell deficiency (LSCD) is a pathologic condition caused by the dysfunction and/or destruction of stem cell precursors of the corneal epithelium, typified clinically by corneal conjunctivalization. The purpose of this review is to critically discuss a less well-known cause of limbal stem cell disease: contact lens (CL) wear. A literature search was conducted to include original articles containing patients with CL-induced LSCD. This review describes epidemiology, diagnostic strategies, pathogenesis, differential diagnosis, and treatment modalities for this condition.

  20. Corneal epithelial stem cells: deficiency and regulation.

    PubMed

    Secker, Genevieve A; Daniels, Julie T

    2008-09-01

    The corneal epithelium is continuously renewed by a population of stem cells that reside in the corneoscleral junction, otherwise known as the limbus. These limbal epithelial stem cells (LESC) are imperative for corneal maintenance with deficiencies leading to in-growth of conjunctival cells, neovascularisation of the corneal stroma and eventual corneal opacity and visual loss. One such disease that has traditionally been thought to be due to LESC deficiency is aniridia, a pan-ocular congenital eye disease due to mutations in the PAX6 gene. Corneal changes or aniridia related keratopathy (ARK) seen in aniridia are typical of LESC deficiency. However, the pathophysiology behind ARK is still ill defined, with current theories suggesting it may be caused by a deficiency in the stem cell niche and adjacent corneal stroma, with altered wound healing responses also playing a role (Ramaesh et al, International Journal of Biochemistry & Cell Biology 37:547-557, 2005) or abnormal epidermal differentiation of LESC (Li et al., The Journal of Pathology 214:9, 2008). PAX6 is considered the master control gene for the eye and is required for normal eye development with expression continuing in the adult cornea, thus inferring a role for corneal repair and regeneration (Sivak et al., Developments in Biologicals 222:41-54, 2000). Studies of models of Pax6 deficiency, such as the small eyed (sey) mouse, should help to reveal the intrinsic and extrinsic mechanisms involved in normal LESC function.

  1. STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION

    PubMed Central

    Loewenstein, Werner R.; Kanno, Yoshinobu

    1964-01-01

    Membrane permeability of an epithelial cell junction (Drosophila salivary gland) was examined with intracellular microelectrodes and with fluorescent tracers. In contrast to the non-junctional cell membrane surface, which has a low permeability to ions (10-4 mho/cm2), the junctional membrane surface is highly permeable. In fact, it introduces no substantial restriction to ion flow beyond that in the cytoplasm; the resistance through a chain of cells (150 Ω cm) is only slightly greater than in extruded cytoplasm (100 Ω cm). The diffusion resistance along the intercellular space to the exterior, on the other hand, is very high. Here, there exists an ion barrier of, at least, 104Ω cm2. As a result, small ions and fluorescein move rather freely from one cell to the next, but do not leak appreciably through the intercellular space to the exterior. The organ here, rather than the single cell, appears to be the unit of ion environment. The possible underlying structural aspects are discussed. PMID:14206423

  2. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  3. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  4. Unique and Analogous Functions of Aquaporin 0 for Fiber Cell Architecture and Ocular Lens Transparency

    PubMed Central

    Kumari, S. Sindhu; Eswaramoorthy, Subramaniam; Mathias, Richard T.; Varadaraj, Kulandaiappan

    2011-01-01

    Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein. To validate this notion, we determined if wild type (WT) lens ultrastructure and fiber cell adhesion are different in AQP0−/−, and TgAQP1+/+/AQP0−/− mice that transgenically express AQP1 (TgAQP1) in fiber cells without AQP0 (AQP0−/−). In WT, lenses were transparent with ‘Y’ sutures. Fibers contained opposite end curvature, lateral interdigitations and hexagonal shape, and were arranged as concentric growth shells. AQP0−/− lenses were cataractous, lacked ‘Y’ sutures, ordered packing and well-defined lateral interdigitations. TgAQP1+/+/AQP0−/− lenses showed improvement in transparency and lateral interdigitations in the outer cortex while inner cortex and nuclear fibers were severely disintegrated. Transmission electron micrographs exhibited tightly packed fiber cells in WT whereas AQP0−/− and TgAQP1+/+/AQP0−/− lenses had wide extracellular spaces. Fibers were easily separable by teasing in AQP0−/− and TgAQP1+/+/AQP0−/− lenses compared to WT. Our data suggest that the increased water permeability through AQP1 does not compensate for loss of AQP0 expression in TgAQP1+/+/AQP0−/− mice. Fiber cell AQP0 expression is required to maintain their organization, which is a requisite for lens transparency. AQP0 appears necessary for cell-to-cell adhesion and thereby to minimize light scattering since in the AQP0−/− and TgAQP1+/+/AQP0−/− lenses, fiber cell disorganization was evident. PMID:21511033

  5. Multi-functionality and plasticity characterize epithelial cells in Hydra

    PubMed Central

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  6. N-myc regulates growth and fiber cell differentiation in lens development

    PubMed Central

    Cavalheiro, Gabriel R.; Matos-Rodrigues, Gabriel E.; Zhao, Yilin; Gomes, Anielle L.; Anand, Deepti; Predes, Danilo; de Lima, Silmara; Abreu, Jose G.; Zheng, Deyou; Lachke, Salil A.; Cvekl, Ales; Martins, Rodrigo A. P.

    2017-01-01

    Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc--deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc--deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIβ. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis. PMID:28716713

  7. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis.

    PubMed

    Ewald, Andrew J; Brenot, Audrey; Duong, Myhanh; Chan, Bianca S; Werb, Zena

    2008-04-01

    Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly, suggesting common mechanisms of epithelial growth.

  8. Osteopontin traffic in hypoxic renal epithelial cells.

    PubMed

    Hampel, Dierk J; Sansome, Christine; Romanov, Victor I; Kowalski, Aaron J; Denhardt, David T; Goligorsky, Michael S

    2003-01-01

    Osteopontin (OPN), a secretory RGD-containing phosphoprotein, is induced in acute renal injury where it plays a renoprotective role. To investigate in depth the mode of OPN secretion under stress conditions, we analyzed OPN traffic in human renal proximal tubular epithelial cells (RPTEC). Western blot analysis and fluorescence microscopy revealed trace amounts of OPN in intact cells, whereas cytoplasmic OPN levels were significantly increased after 24-48 h hypoxia. Immunoelectron microscopy of RPTEC showed predominantly apical localization of gold-labeled OPN under normal conditions. Hypoxia (24 h) increased 2.5-fold immunodetectable gold-labeled OPN at the apical plasma membrane; further reoxygenation (2 h) augmented apical and basolateral labeling 2- and 10-fold, respectively. Analysis of apical and basolateral medium conditioned by RPTEC grown on semipermeable membranes using a specially developed ELISA showed a global decrease in secreted OPN after hypoxia, which recovered following 2 h reoxygenation. Agents known to disrupt the function of the Golgi apparatus (brefeldin A, monensin) or actin cytoskeleton (cytochalasin B) significantly inhibited OPN-GFP secretion in normoxic cells. In cells recovering from hypoxia, however, OPN secretion required functional Golgi apparatus, but was not affected by cytochalasin B. These findings demonstrate that stress inhibits OPN secretion by the process dependent on the functional Golgi apparatus and actin cytoskeleton; recovery restores OPN secretion, although its polarity may become perturbed. Copyright 2003 S. Karger AG, Basel

  9. Identification and Characterization of FGF2-Dependent mRNA: microRNA Networks During Lens Fiber Cell Differentiation

    PubMed Central

    Wolf, Louise; Gao, Chun S.; Gueta, Karen; Xie, Qing; Chevallier, Tiphaine; Podduturi, Nikhil R.; Sun, Jian; Conte, Ivan; Zelenka, Peggy S.; Ashery-Padan, Ruth; Zavadil, Jiri; Cvekl, Ales

    2013-01-01

    MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation. PMID:24142921

  10. Seeding of recipient-originated epithelial cells attenuates epithelial to mesenchymal transition in rat tracheal allotransplantation.

    PubMed

    Huang, Xun; Yan, Xiaolong; Zhang, Zhipei; Li, Xiaofei

    2015-06-01

    The specific role and mechanism of epithelium in the progression of obliterative airway disease (OAD) after tracheal allotransplantation remain poorly understood. In this study, we used rat heterotopic tracheal transplantation to investigate the mechanism of epithelial cell seeding during the process of OAD. Prospective, basic science. Research laboratory. In total, 120 Sprague Dawley (SD) rats and 90 Wistar rats were used. Tracheas from SD rats were implanted into SD rats (syngeneic, n = 30) or Wistar rats (allogeneic, n = 30), and SD rat tracheas (seeded with Wistar rat-derived epithelial cells 6 days after transplantation) were transplanted into Wistar rats (seeded allogeneic, n = 30). Grafts were harvested at 7, 14, or 30 days after transplantation for histologic, quantitative reverse transcriptional polymerase chain reaction or Western blot analyses. Syngrafts retained normal histologic structures, while the corresponding allografts demonstrated less ciliated epithelia and more lumenal occlusion. Seeding of epithelial cells ameliorated the histologic changes, reduced the expression of epithelial to mesenchymal transition (EMT)-related transcriptional factors and mesenchymal markers, and dampened the expression of transforming growth factor β1 (TGF-β1) and phosphorylation of smad3. Seeding of recipient epithelial cells inhibits the progression of OAD by attenuating EMT via TGF-β-Smad signaling in rat heterotopic tracheal allografts. Clinically, the injection of recipient-originated epithelial cells might provide new insights into the treatment for OAD after tracheal allotransplantation. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

  11. Cytomatrix synthesis in MDCK epithelial cells

    SciTech Connect

    Mitchell, J.J.; Low, R.B.; Woodcock-Mitchell, J.L. )

    1990-06-01

    Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with (14C)leucine over several days and then pulse-labeled for 4 hours with (3H)leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form.

  12. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

  13. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  14. The cause and consequence of fiber cell compaction in the vertebrate lens

    PubMed Central

    Bassnett, Steven; Costello, M. Joseph

    2016-01-01

    Fiber cells of the ocular lens are arranged in a series of concentric shells. New growth shells are added continuously to the lens surface and, as a consequence, the preexisting shells are buried. To focus light, the refractive index of the lens cytoplasm must exceed that of the surrounding aqueous and vitreous humors, and to that end, lens cells synthesize high concentrations of soluble proteins, the crystallins. To correct for spherical aberration, it is necessary that the crystallin concentration varies from shell-to-shell, such that cellular protein content is greatest in the center of the lens. The radial variation in protein content underlies the critical gradient index (GRIN) structure of the lens. Only the outermost shells of lens fibers contain the cellular machinery necessary for protein synthesis. It is likely, therefore, that the GRIN (which spans the synthetically inactive, organelle-free zone of the lens) does not result from increased levels of protein synthesis in the core of the lens but is instead generated through loss of volume by inner fiber cells. Because volume is lost primarily in the form of cell water, the residual proteins in the central lens fibers can be concentrated to levels of >500 mg/ml. In this short review, we describe the process of fiber cell compaction, its relationship to lens growth and GRIN formation, and offer some thoughts on the likely nature of the underlying mechanism. PMID:26992780

  15. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

    PubMed

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-07-15

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

  16. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

    PubMed Central

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-01-01

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite. PMID:25221599

  17. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  18. Cell death at the intestinal epithelial front line.

    PubMed

    Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

    2016-07-01

    The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

  19. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    PubMed Central

    Rodrigues-Diez, Raquel; Rodrigues-Diez, Raúl R.; Lavoz, Carolina; Carvajal, Gisselle; Droguett, Alejandra; Garcia-Redondo, Ana B.; Rodriguez, Isabel; Ortiz, Alberto; Egido, Jesús; Mezzano, Sergio; Ruiz-Ortega, Marta

    2014-01-01

    Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β. PMID:24949470

  20. Epithelial-to-mesenchymal transition in penile squamous cell carcinoma.

    PubMed

    Masferrer, Emili; Ferrándiz-Pulido, Carla; Masferrer-Niubò, Magalí; Rodríguez-Rodríguez, Alfredo; Gil, Inmaculada; Pont, Antoni; Servitje, Octavi; García de Herreros, Antonio; Lloveras, Belen; García-Patos, Vicenç; Pujol, Ramon M; Toll, Agustí; Hernández-Muñoz, Inmaculada

    2015-02-01

    Epithelial-to-mesenchymal transition is a phenomenon in epithelial tumors that involves loss of intercellular adhesion, mesenchymal phenotype acquisition and enhanced migratory potential. While the epithelial-to-mesenchymal transition process has been extensively linked to metastatic progression of squamous cell carcinoma, studies of the role of epithelial-to-mesenchymal transition in squamous cell carcinoma containing high risk human papillomaviruses are scarce. Moreover, to our knowledge epithelial-to-mesenchymal transition involvement in human penile squamous cell carcinoma, which can arise through transforming HPV infections or independently of HPV, has not been investigated. We evaluated the presence of epithelial-to-mesenchymal transition markers and their relationship to HPV in penile squamous cell carcinoma. We assessed the expression of E-cadherin, vimentin and the epithelial-to-mesenchymal transition related transcription factors Twist, Zeb1 and Snail by immunohistochemical staining in 64 penile squamous cell carcinoma cases. HPV was detected by polymerase chain reaction amplification. Simultaneous loss of membranous E-cadherin expression and vimentin over expression were noted in 43.5% of penile squamous cell carcinoma cases. HPV was significantly associated with loss of membranous E-cadherin but not with epithelial-to-mesenchymal transition. Recurrence and mortality rates were significantly higher in cases showing epithelial-to-mesenchymal transition. Our findings indicate that in penile squamous cell carcinoma epithelial-to-mesenchymal transition is associated with poor prognosis but not with the presence of HPV. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  1. Clinical implications of epithelial cell plasticity in cancer progression.

    PubMed

    Aparicio, Luis A; Blanco, Moisés; Castosa, Raquel; Concha, Ángel; Valladares, Manuel; Calvo, Lourdes; Figueroa, Angélica

    2015-09-28

    In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients.

  2. Human Growth Hormone Promotes Corneal Epithelial Cell Migration in Vitro

    PubMed Central

    Ding, Juan; Wirostko, Barbara; Sullivan, David A

    2015-01-01

    Purpose Corneal wound healing is a highly regulated process that requires the proliferation and migration of epithelial cells and interactions between epithelial cells and stromal fibroblasts. Compounds that can be applied topically to the ocular surface and that have the capability of activating corneal epithelial cells to proliferate and/or migrate would be useful to promote corneal wound healing. We hypothesize that human growth hormone (HGH) will activate Signal Transducer and Activators of Transcription-5 (STAT5) signaling and promote corneal wound healing by enhancing corneal epithelial cell and fibroblast proliferation and/or migration in vitro. The purpose of this study is to test these hypotheses. Methods We studied cell signaling, proliferation and migration using an immortalized human corneal epithelial cell line and primary human corneal fibroblasts in vitro. We also examined whether insulin-like growth factor-1 (IGF-1), a hormone known to mediate many of HGH’s growth promoting actions, may play a role in this effect. Results We show that HGH activates STAT5 signaling and promotes corneal epithelial cell migration in vitro. The migratory effect requires an intact communication between corneal epithelia and fibroblasts, and is not mediated by IGF-1. Conclusion HGH may represent a topical therapeutic to promote corneal epithelial wound healing. This warrants further investigation. PMID:25782399

  3. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration.

    PubMed

    Kumar, J Dinesh; Steele, Islay; Moore, Andrew R; Murugesan, Senthil V; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea; Dockray, Graham J

    2015-07-15

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling.

  4. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  5. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  6. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

    This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

  7. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

  8. Cultured epithelial cells response to phototherapy with low intensity laser.

    PubMed

    Eduardo, Fernanda P; Mehnert, Dolores U; Monezi, Telma A; Zezell, Denise M; Schubert, Mark M; Eduardo, Carlos P; Marques, Márcia M

    2007-04-01

    Little is known about the intracellular response of epithelial cells to phototherapy. The aim of this in vitro study was to analyze the effect of phototherapy with low-energy lasers with different wavelengths and powers on cultured epithelial cell growth under different nutritional conditions. Epithelial cell cultures (Vero cell line) grown in nutritional deficit in culture medium supplemented with 2% fetal bovine serum (FBS) were irradiated with low-energy laser from one to three times with a GaAlAs laser (660 nm) and InGaAlP (780 nm), 40 and 70 mW, respectively, with 3 or 5 J/cm2. Cell growth was indirectly assessed by measuring the cell mitochondrial activity. Nonirradiated cell cultures grown in nutritional regular medium supplemented with 10% FBS produced higher cell growth than all cultures grown in nutritional deficit irradiated or not. The overall cell growth of cultures grown under nutritionally deficit conditions was significantly improved especially when irradiated with 780 nm for three times. Phototherapy with the laser parameters tested increases epithelial cell growth rate for cells stressed by growth under nutritionally deficient states. This cell growth improvement is directly proportional to the number of irradiations; however, was not enough to reach the full cell growth potential rate of Vero epithelial cell line observed when growing under nutritional regular condition. (c) 2007 Wiley-Liss, Inc.

  9. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  10. Dual Roles for Pax-6: a Transcriptional Repressor of Lens Fiber Cell-Specific β-Crystallin Genes

    PubMed Central

    Duncan, Melinda K.; Haynes, John I.; Cvekl, Ales; Piatigorsky, Joram

    1998-01-01

    It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the αB-, αA-, δ1-, and ζ-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and β-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the βB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed βB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (−183 to −152, −120 to −48, and −30 to +1) of the βB1-crystallin promoter. Earlier experiments showed that the βB1-crystallin promoter sequence from −120 to −48 contains a cis element (PL2 at −90 to −76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the βB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of βB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens

  11. HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

    PubMed Central

    Tugizov, Sharof M.; Herrera, Rossana; Veluppillai, Piri; Greenspan, Deborah; Soros, Vanessa; Greene, Warner C.; Levy, Jay A.; Palefsky, Joel M.

    2010-01-01

    Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission. PMID:21056450

  12. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Eule, Stephan; Metzger, Jakob; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Grosshans, Joerg; Wolf, Fred

    2015-03-01

    We found that the constriction of epithelial cell contacts during intercalation in germ band extension in Drosophila embryos follows intriguingly simple quantitative laws. The mean contact length < L > follows < L > (t) ~(T - t) α , where T is the finite collapse time; the time dependent variance of contact length is proportional to the square of the mean; finally the time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations suggest that the dynamics of contact collapse can be captured by a stochastic differential equation analytically tractable in small noise approximation. Here, we present such a model, providing an effective description of the non-equilibrium statistical mechanics of contact collapse. All model parameters are fixed by measurements of time dependent mean and variance of contact lengths. The model predicts the contact length covariance function that we obtain in closed form. The contact length covariance function closely matches experimental observations suggesting that the model well captures the dynamics of contact collapse.

  13. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  14. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  15. Culture, Immortalization, and Characterization of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Liu, Shaohui; Hatton, Mark P.; Khandelwal, Payal

    2010-01-01

    Purpose. Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. Methods. Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. Results. It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. Conclusions. The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized. PMID:20335607

  16. Differentiation State-Specific Mitochondrial Dynamic Regulatory Networks Are Revealed by Global Transcriptional Analysis of the Developing Chicken Lens

    PubMed Central

    Chauss, Daniel; Basu, Subhasree; Rajakaruna, Suren; Ma, Zhiwei; Gau, Victoria; Anastas, Sara; Brennan, Lisa A.; Hejtmancik, J. Fielding; Menko, A. Sue; Kantorow, Marc

    2014-01-01

    The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells. PMID:24928582

  17. Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia

    PubMed Central

    Sugiyama, Yuki; Stump, Richard J. W.; Nguyen, Anke; Wen, Li; Chen, Yongjuan; Wang, Yanshu; Murdoch, Jennifer N.; Lovicu, Frank J.; McAvoy, John W.

    2009-01-01

    Planar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior-posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having a apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial-anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2, shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles. PMID:19968984

  18. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  19. Cell volume regulation in epithelial physiology and cancer

    PubMed Central

    Pedersen, Stine F.; Hoffmann, Else K.; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed. PMID:24009588

  20. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  1. Ion pump sorting in polarized renal epithelial cells.

    PubMed

    Caplan, M J

    2001-08-01

    The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

  2. Secretion of alpha 1-antitrypsin by alveolar epithelial cells.

    PubMed

    Venembre, P; Boutten, A; Seta, N; Dehoux, M S; Crestani, B; Aubier, M; Durand, G

    1994-06-13

    We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce alpha 1-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.

  3. Metformin inhibits the proliferation of benign prostatic epithelial cells

    PubMed Central

    Ge, Rongbin; Li, Jijun; Johnson, Cameron W.; Rassoulian, Cyrus; Olumi, Aria F.

    2017-01-01

    Objective Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the

  4. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    SciTech Connect

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  5. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    SciTech Connect

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with {sup 3}H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-{beta} did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 {times} 10{sup 6} sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined.

  6. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  7. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-12-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction.

  8. Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

    PubMed

    Christopher, Karen L; Pedler, Michelle G; Shieh, Biehuoy; Ammar, David A; Petrash, J Mark; Mueller, Niklaus H

    2014-02-01

    In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro

    PubMed Central

    Zheng, Dahai; Soh, Boon-Seng; Yin, Lu; Hu, Guangan; Chen, Qingfeng; Choi, Hyungwon; Han, Jongyoon; Chow, Vincent T. K.; Chen, Jianzhu

    2017-01-01

    Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells’ capacity to differentiate into alveolar epithelial cells at the single-cell level. PMID:28128362

  10. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

  11. Msx2 plays a critical role in lens epithelium cell cycle control

    PubMed Central

    Zhao, Jiang-Yue; Zhuang, Feng-Feng; Wang, Hong-Yan; Wu, Di; Zhang, Jin-Song

    2013-01-01

    AIM To investigate the effects of Msx2 on lens epithelium cell cycle, and evaluate the changes of the proliferation, apoptosis of lens epithelium cells. METHODS Mice lens epithelium cells were cultured and transfected with pEGFP-Msx2 and control. Msx2-deficient mice (Msx2−/−) lens tissue were isolated. Lens tissue and transfected cells were prepared for mRNA extraction using Trizol reagent. CyclinD1 and Prox1 expression were evaluated by real-time RT-PCR. BrdU incorporation and apoptosis rate were investigated by immunofluorescence and flow cytometry analysis. RESULTS After transfected with pEGFP-Msx2, lens epithelium cells failed to incorporate BrdU and anti-phospho-histone-3 immunofluorescence failed to detect cell nuclei which GFP were positive. Msx2 over expression resulted in increasing apoptosis rate in lens epithelium cells. CyclinD1 and Prox1 expression increased significantly in Msx2 knockout mice by real-time RT-PCR quantization and CyclinD1 expression decreased significantly in Msx2 overexpressed cell. CONCLUSION Msx2 has the effect of inhibiting proliferation and differentiation, triggering apoptosis on mice lens epithelium cells. PMID:23826518

  12. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

    PubMed Central

    Hong, Deli; Messier, Terri L.; Tye, Coralee E.; Dobson, Jason R.; Fritz, Andrew J.; Sikora, Kenneth R.; Browne, Gillian; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

    2017-01-01

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression. PMID:28407681

  13. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition.

    PubMed

    Hong, Deli; Messier, Terri L; Tye, Coralee E; Dobson, Jason R; Fritz, Andrew J; Sikora, Kenneth R; Browne, Gillian; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2017-03-14

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

  14. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration

    PubMed Central

    Kumar, J. Dinesh; Steele, Islay; Moore, Andrew R.; Murugesan, Senthil V.; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D. Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea

    2015-01-01

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling. PMID:25977510

  15. DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

    PubMed

    Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

    2014-04-01

    DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

  16. Anatomical location and culture of equine corneal epithelial stem cells.

    PubMed

    Moriyama, Hidekazu; Kasashima, Yoshinori; Kuwano, Atsutoshi; Wada, Shinya

    2014-03-01

    To identify morphologically the locations of equine corneal epithelial stem cells (CESCs) and to culture these cells. We studied the eyes of 12 adult thoroughbred horses. Eye tissues were immunostained for two positive stem cell markers (p63, CK14) and one negative marker (CK3) to identify the locations of CESCs, so we could compare their immunostaining patterns with those of human stem cells previously reported. We compared the proliferation rates and morphological features of epithelial cells isolated from the corneal limbus and central cornea. Undifferentiated cells expressing the same immunostaining pattern as human CESCs were present in the equine corneal limbus. Cultured epithelial cells isolated from the limbus expressed the same immunostaining pattern that CESCs show histologically, but cells isolated from the central cornea did not proliferate and could not be evaluated. Equine CESCs were localized in the epithelial basal layer of the corneal limbus, where melanocytes reside. They could be cultured without loss of their undifferentiated nature. When collecting such stem cells, it may be useful to harvest and culture corneal epithelial tissues in the limbus where melanocytes serve as an indicator of the collecting area. © 2013 American College of Veterinary Ophthalmologists.

  17. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  18. Epithelial to Mesenchymal Transition of Mesothelial Cells in Tuberculous Pleurisy

    PubMed Central

    Kim, Changhwan; Park, Sung-Hoon; Hwang, Yong Il; Jang, Seung Hun; Kim, Cheol Hong; Jung, Ki-Suck; Min, Kwangseon; Lee, Jae Woong; Jang, Young Sook

    2011-01-01

    Purpose Tuberculous pleurisy is the most frequent extrapulmonary manifestation of tuberculosis. In spite of adequate treatment, pleural fibrosis is a common complication, but the mechanism has not been elucidated. This study is to determine whether epithelial to mesenchymal transition (EMT) of mesothelial cells occurs in tuberculous pleurisy. Materials and Methods Normal pleural mesothelial cells, isolated from irrigation fluids during operations for primary spontaneous pneumothorax, were characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). These cells were treated in vitro with various cytokines, which were produced in the effluents of tuberculous pleurisy. The isolated cells from the effluents of tuberculous pleurisy were analyzed by immunofluorescence and RT-PCR analysis. Results The isolated cells from the irrigation fluid of primary spontaneous pneumothorax had epithelial characteristics. These cells, with transforming growth factor-β1 and/or interleukin-1β treatment, underwent phenotypic transition from epithelial to mesenchymal cells, with the loss of epithelial morphology and reduction in cytokeratin and E-cadherin expression. Effluent analysis from tuberculous pleurisy using immunofluorescence and RT-PCR demonstrated two phenotypes that showed mesenchymal characteristics and both epithelial & mesencymal characteristics. Conclusion Our results suggest that pleural mesothelial cells in tuberculous pleurisy have been implicated in pleural fibrosis through EMT. PMID:21155035

  19. Epithelial cell guidance by self-generated EGF gradients†

    PubMed Central

    Scherber, Cally; Aranyosi, Alexander J.; Kulemann, Birte; Thayer, Sarah P.; Toner, Mehmet; Iliopoulos, Othon

    2012-01-01

    Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing. PMID:22314635

  20. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  1. Starved epithelial cells uptake extracellular matrix for survival

    PubMed Central

    Muranen, Taru; Iwanicki, Marcin P.; Curry, Natasha L.; Hwang, Julie; DuBois, Cory D.; Coloff, Jonathan L.; Hitchcock, Daniel S.; Clish, Clary B.; Brugge, Joan S.; Kalaany, Nada Y.

    2017-01-01

    Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition. PMID:28071763

  2. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  3. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  4. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  5. Development of human epithelial cell systems for radiation risk assessment

    NASA Astrophysics Data System (ADS)

    Yang, C. H.; Craise, L. M.

    1994-10-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  6. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium.

    PubMed

    Vasavada, A R; Thampi, P; Yadav, S; Rawal, U M

    1993-12-01

    The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

  7. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling

    PubMed Central

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. DOI: http://dx.doi.org/10.7554/eLife.15034.001 PMID:27431614

  8. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

  9. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

    Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs. PMID:23241529

  10. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

  11. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  12. Mechanobiology in Lung Epithelial Cells: Measurements, Perturbations, and Responses

    PubMed Central

    Waters, Christopher M.; Roan, Esra; Navajas, Daniel

    2015-01-01

    Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis. PMID:23728969

  13. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    PubMed

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  14. Epithelial-mesenchymal transitions of bile duct epithelial cells in primary hepatolithiasis.

    PubMed

    Zhao, Lijin; Yang, Rigao; Cheng, Long; Wang, Maijian; Jiang, Yan; Wang, Shuguang

    2010-07-01

    The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, alpha-catenin, alpha-SMA, vimentin, S100A4, TGF-beta1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and alpha-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, alpha-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-beta1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-beta1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.

  15. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  16. The nuclear envelope in the crystalline lens fiber cell.

    PubMed

    Harding, C V; Susan, S R

    1976-05-01

    Rabbit lenses which have been fixed, dehydrated, and dried by a critical-point drying method, can be fractured through the cytoplasm of the differentiating lens fibers, exposing the cell nuclei. The fracture, under these conditions, causes a complete separation of the two membranes of the nuclear envelope from one another, thus exposing entire membrane surfaces (those which line the perinuclear space). These surfaces are not seen in their entirety in typical freeze-fracture or freeze-etch preparations, and consequently have not been described previously. The exposed membrane surfaces which line the perinuclear space have numerous convex structures of approximately 1,000 A, and some larger more irregularly shaped structures. These appear to be fragments of the nuclear pore complexes. Differences in these structures between young fibers and those nearing completion of differentiation is suggested.

  17. Fibrin glue inhibits migration of ocular surface epithelial cells.

    PubMed

    Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

    2016-10-01

    PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

  18. Serratia marcescens internalization and replication in human bladder epithelial cells

    PubMed Central

    Hertle, Ralf; Schwarz, Heinz

    2004-01-01

    Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

  19. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    PubMed

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  20. Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture.

    PubMed

    Chen, Yongjuan; Stump, Richard J W; Lovicu, Frank J; Shimono, Akihiko; McAvoy, John W

    2008-12-01

    How an organ develops its characteristic shape is a major issue. This is particularly critical for the eye lens as its function depends on having appropriately ordered three-dimensional cellular architecture. Recent in vitro studies indicate that Wnt signaling plays key roles in regulating morphological events in FGF-induced fiber cell differentiation in the mammalian lens. To further investigate this the Wnt signaling antagonist, secreted frizzled-related protein 2 (Sfrp2), was overexpressed in lens fiber cells of transgenic mice. In these mice fiber cell elongation was attenuated and individual fibers exhibited irregular shapes and consequently did not align or pack regularly; microtubules, microfilaments and intermediate filaments were clearly disordered in these fibers. Furthermore, a striking feature of transgenic lenses was that fibers did not develop the convex curvature typically seen in normal lenses. This appears to be related to a lack of protrusive processes that are required for directed migratory activity at their apical and basal tips as well as for the formation of interlocking processes along their lateral margins. Components of the Wnt/Planar Cell Polarity (PCP) pathway were downregulated or inhibited. Taken together this supports a role for Wnt/PCP signaling in orchestrating the complex organization and dynamics of the fiber cell cytoskeleton.

  1. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

    PubMed

    Allahverdian, Sima; Harada, Norihiro; Singhera, Gurpreet K; Knight, Darryl A; Dorscheid, Delbert R

    2008-02-01

    Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

  2. Biphenotypic surface epithelial cells in the gastrointestinal tube with mixed epithelial-myofibroblastic differentiation: a paradigm.

    PubMed

    Németh, István Balázs; Tiszlavicz, László

    2012-04-01

    Epithelial cells and myofibroblasts are well-characterized histomorphological elements of tissues. They are distinguished from one another on the basis of topography and of differences in cytokeratin (CK) and α-smooth muscle actin (SMA) expression. Certain epithelial cells exhibit CK / SMA co-expression. This study aimed to define the immunophenotypical characteristics of these biphenotypic cells with respect to cytodifferentiation (broad spectrum of CKs, SMA), cell-cell interaction (E-cadherin, adenomatous polyposis coli - APC, β-catenin), and cell survival (cyclooxygenase-2 - Cox-2). At the routine gastrointestinal pathology service of the Department of Pathology, University of Szeged, tissue samples were identified from instances of cervical inlet patch (n = 5), Barrett's esophagus (n=5), gastritis (n=5), fundic gland polyp (n=2), gastric neoplastic polyp (n=1), inflammatory bowel disease (n=5), and colonic neoplastic polyp (n=3). that contained epithelial cells expressing SMA. These biphenotypic cells were further immunophenotyped. Foregut-derived biphenotypic cells expressed CKs 7 and 20, while hindgut-derived biphenotypic cells expressed only CK 20. Subepithelial myofibroblasts adjacent to biphenotypic epithelium expressed Cox-2, SMA, and β-catenin, as did biphenotypic cells. Myofibroblasts, however, did not express CKs. In neoplastic polyps, APC expression weakened as cytologic atypism increased, while intermingled biphenotypic cells in neoplastic glands overexpressed APC, as did myofibroblasts beneath. CK subspecies expression in biphenotypic cells reflects embryonic development of the gastrointestinal tract. The immunophenotyping analysis addresses bidirectional (via transdifferentiation from epithelia into myofibroblasts or vice versa) formation of biphenotypic cells within damaged epithelium, a phenomenon that must be further analysed.

  3. Expression of pluripotency factors in larval epithelia of the frog Xenopus: Evidence for the presence of cornea epithelial stem cells

    PubMed Central

    Perry, Kimberly J.; Thomas, Alvin G.; Henry, Jonathan J.

    2013-01-01

    cornea. Using a thymidine analog (EdU), we were able to label mitotically active cells, which revealed that cell proliferation takes place throughout the cornea epithelium, predominantly in the basal epithelial layer. Species of Xenopus and one other amphibian are unique in their ability to replace a missing lens from cells derived from the basal cornea epithelium. Using EdU we show, as others have previously, that proliferating cells within the cornea epithelium do contribute to the formation of these regenerated lenses. Furthermore, using qPCR we determined that representatives of various pluripotency genes (i.e., sox2, p63 and oct60) are upregulated early during the process of lens regeneration. Antibody labeling showed that the number of sox2 expressing cells increased dramatically within 4 hours following lens removal and these cells were scattered throughout the basal layer of the cornea epithelium. Historically, the process of lens regeneration in Xenopus had been described as one involving transdifferentiation of cornea epithelial cells (i.e., one involving cellular dedifferentiation followed by redifferentiation). Our combined observations provide evidence that a population of stem cells exists within the Xenopus cornea. We hypothesize that the basal epithelium contains oligopotent epithelial stem cells that also represent the source of regenerated lenses in the frog. Future studies will be required to clearly identify the source of these lenses. PMID:23274420

  4. Expression of pluripotency factors in larval epithelia of the frog Xenopus: evidence for the presence of cornea epithelial stem cells.

    PubMed

    Perry, Kimberly J; Thomas, Alvin G; Henry, Jonathan J

    2013-02-15

    cornea. Using a thymidine analog (EdU), we were able to label mitotically active cells, which revealed that cell proliferation takes place throughout the cornea epithelium, predominantly in the basal epithelial layer. Species of Xenopus and one other amphibian are unique in their ability to replace a missing lens from cells derived from the basal cornea epithelium. Using EdU we show, as others have previously, that proliferating cells within the cornea epithelium do contribute to the formation of these regenerated lenses. Furthermore, using qPCR we determined that representatives of various pluripotency genes (i.e., sox2, p63 and oct60) are upregulated early during the process of lens regeneration. Antibody labeling showed that the number of sox2 expressing cells increased dramatically within 4 h following lens removal and these cells were scattered throughout the basal layer of the cornea epithelium. Historically, the process of lens regeneration in Xenopus had been described as one involving transdifferentiation of cornea epithelial cells (i.e., one involving cellular dedifferentiation followed by redifferentiation). Our combined observations provide evidence that a population of stem cells exists within the Xenopus cornea. We hypothesize that the basal epithelium contains oligopotent epithelial stem cells that also represent the source of regenerated lenses in the frog. Future studies will be required to clearly identify the source of these lenses. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    PubMed

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

  6. Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

    PubMed

    Becker, Stephen M; Cho, Kyou-Nam; Guo, Xiaoti; Fendig, Kirsten; Oosman, Mohammed N; Whitehead, Robert; Cohn, Steven M; Houpt, Eric R

    2010-03-01

    Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

  7. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells.

    PubMed

    Bosshardt, D D; Nanci, A

    1998-02-01

    After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

  8. Dual function of Yap in the regulation of lens progenitor cells and cellular polarity.

    PubMed

    Song, Ji Yun; Park, Raehee; Kim, Jin Young; Hughes, Lucinda; Lu, Li; Kim, Seonhee; Johnson, Randy L; Cho, Seo-Hee

    2014-02-15

    Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, β-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.

  9. Dendritic immune cell densities in the central cornea associated with soft contact lens types and lens care solution types: a pilot study

    PubMed Central

    Sindt, Christine W; Grout, Trudy K; Critser, D Brice; Kern, Jami R; Meadows, David L

    2012-01-01

    Background The purpose of this study was to assess whether differences in central corneal dendritic immune cell densities associated with combinations of soft contact lenses and lens care solutions could be detected by in vivo confocal microscopy. Methods Participants were adults naïve to contact lens wear (n = 10) or who wore soft contact lenses habitually on a daily-wear schedule (n = 38) or on a study-assigned schedule for 30 days with daily disposable silicone hydrogel lenses (n = 15). Central corneas were scanned using an in vivo confocal microscope. Cell densities were compared among groups by demographic parameters, lens materials, and lens care solutions (polyhexamethylene biguanide [PHMB], polyquaternium-1 and myristamidopropyl dimethylamine [PQ/MAPD], peroxide, or blister pack solution [for daily disposable lenses]). Results Among lens wearers, no associations were observed between immune cell densities and age, gender, or years of lens-wearing experience. Mean cell density was significantly lower (P < 0.01) in nonwearers (29 ± 23 cells/mm2, n = 10) than in lens wearers (64 ± 71 cells/mm2, n = 53). Mean cell density was lower (P = 0.21) with traditional polymer lenses (47 ± 44 cells/mm2, n = 12) than with silicone hydrogel lenses (69 ± 77 cells/mm2, n = 41). Lowest to highest mean density of immune cells among lens wearers was as follows: PQ/MAPD solution (49 ± 28 cells/mm2), blister pack solution (63 ± 81 cells/mm2), PHMB solution (66 ± 44