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  1. 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

    2016-01-01

    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. PMID:27431614

  2. 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

  3. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi

    PubMed Central

    Val, Stéphanie; Burgett, Katelyn; Brown, Kristy J.; Preciado, Diego

    2016-01-01

    Background Chronic Otitis Media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute Otitis Media (OM) pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line. Methods NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC) cultured at air-liquid interface over 48 hours– 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling. Results Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05). The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface. Conclusions NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level. PMID:26859300

  4. Vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells

    PubMed Central

    Rogel, Micah R.; Soni, Pritin N.; Troken, James R.; Sitikov, Albert; Trejo, Humberto E.; Ridge, Karen M.

    2011-01-01

    The physiological and pathophysiological implications of the expression of vimentin, a type III intermediate filament protein, in alveolar epithelial cells (AECs) are unknown. We provide data demonstrating that vimentin is regulated by TGFβ1, a major cytokine released in response to acute lung injury and that vimentin is required for wound repair and remodeling of the alveolar epithelium. Quantitative real-time PCR shows a 16-fold induction of vimentin mRNA in TGFβ1-treated transformed AECs. Luciferase assays identify a Smad-binding element in the 5′ promoter of vimentin responsible for TGFβ1-induced transcription. Notably, TGFβ1 induces vimentin protein expression in AECs, which is associated with a 2.5-fold increase in cell motility, resulting in increased rates of migration and wound closure. These effects are independent of cell proliferation. TGFβ1-mediated vimentin protein expression, cell migration, and wound closure are prevented by a pharmacological inhibitor of the Smad pathway and by expression of Ad-shRNA against vimentin. Conversely, overexpression of mEmerald-vimentin is sufficient for increased cell-migration and wound-closure rates. These results demonstrate that vimentin is required and sufficient for increased wound repair in an in vitro model of lung injury.—Rogel, M. R., Soni, P. N., Troken, J. R., Sitikov, A., Trejo, H. E., Ridge, K. M. Vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells. PMID:21803859

  5. Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis

    PubMed Central

    Naikawadi, Ram P.; Disayabutr, Supparerk; Mallavia, Benat; Donne, Matthew L.; Green, Gary; La, Janet L.; Rock, Jason R.; Looney, Mark R.; Wolters, Paul J.

    2016-01-01

    Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin+ mesenchymal cells, collagen deposition, and accumulation of senescence-associated β-galactosidase+ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction. PMID:27699234

  6. Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis

    PubMed Central

    Naikawadi, Ram P.; Disayabutr, Supparerk; Mallavia, Benat; Donne, Matthew L.; Green, Gary; La, Janet L.; Rock, Jason R.; Looney, Mark R.; Wolters, Paul J.

    2016-01-01

    Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin+ mesenchymal cells, collagen deposition, and accumulation of senescence-associated β-galactosidase+ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction.

  7. Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

    PubMed Central

    Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-01-01

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

  8. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.

    PubMed

    Luissint, Anny-Claude; Parkos, Charles A; Nusrat, Asma

    2016-10-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  9. DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

    PubMed Central

    Habibovic, Aida; Hristova, Milena; Heppner, David E.; Danyal, Karamatullah; Ather, Jennifer L.; Janssen-Heininger, Yvonne M.W.; Irvin, Charles G.; Poynter, Matthew E.; Lundblad, Lennart K.; Dixon, Anne E.; Geiszt, Miklos

    2016-01-01

    Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management. PMID:27812543

  10. The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation.

    PubMed

    Coradini, Danila; Oriana, Saro

    2014-02-01

    During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG) and Polycomb (PcG) group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.

  11. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

    PubMed

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A; Wertheimer, Joshua; Mullin, James M

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

  12. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients

    PubMed Central

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A.; Wertheimer, Joshua; Mullin, James M.

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed. PMID:26226276

  13. Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling.

    PubMed

    Boutinaud, Marion; Galio, Laurent; Lollivier, Vanessa; Finot, Laurence; Wiart, Sandra; Esquerré, Diane; Devinoy, Eve

    2013-10-16

    Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

  14. Monocyte interaction accelerates HCl-induced lung epithelial remodeling

    PubMed Central

    2014-01-01

    Background Acute respiratory distress syndrome (ARDS) is characterized by overwhelming inflammatory responses and lung remodeling. We hypothesized that leukocyte infiltration during the inflammatory response modulates epithelial remodeling through a mechanism of epithelial-mesenchymal transition (EMT). Methods Human lung epithelial cells were treated for 30 min with hydrochloric acid (HCl). Human monocytes were then cocultured with the epithelial cells for up to 48 h, in the presence or absence of blocking peptides against lymphocyte function-associated antigen-1 (LFA-1), or tyrphostin A9, a specific inhibitor for platelet-derived growth factor (PDGF) receptor tyrosine kinase. Results Exposure of lung epithelial cells to HCl resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and production of interleukin (IL)-8 at 24 h. The expression of the epithelial markers E-cadherin decreased while the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) increased at 24 h and remained high at 48 h. The addition of monocytes augmented the profiles of lower expression of epithelial markers and higher mesenchymal markers accompanied by increased collagen deposition. This EMT profile was associated with an enhanced production of IL-8 and PDGF. Treatment of the lung epithelial cells with the LAF-1 blocking peptides CD11a237–246 or/and CD18112–122 suppressed monocyte adhesion, production of IL-8, PDGF and hydroxyproline as well as EMT markers. Treatment with tyrphostin A9 prevented the EMT profile shift induced by HCl stimulation. Conclusions The interaction between epithelial cells and monocytes enhanced epithelial remodelling after initial injury through EMT signalling that is associated with the release of soluble mediators, including IL-8 and PDGF. PMID:25108547

  15. YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer

    PubMed Central

    Davies, Alastair H.; Reipas, Kristen M.; Pambid, Mary Rose; Berns, Rachel; Stratford, Anna L.; Fotovati, Abbas; Firmino, Natalie; Astanehe, Arezoo; Hu, Kaiji; Maxwell, Christopher; Mills, Gordon B.; Dunn, Sandra E.

    2015-01-01

    There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated hTERT activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16INK4a-mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors. PMID:24648416

  16. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

    PubMed Central

    Baum, Buzz

    2011-01-01

    The epithelial cadherin (E-cadherin)–catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell’s apical–basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling. PMID:21422226

  17. Remodeling of epithelial cells and basement membranes in a corneal deficiency model with long-term follow-up.

    PubMed

    Kameishi, Sumako; Sugiyama, Hiroaki; Yamato, Masayuki; Sado, Yoshikazu; Namiki, Hideo; Kato, Takashi; Okano, Teruo

    2015-02-01

    The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and

  18. Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

    PubMed Central

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  19. Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism.

    PubMed

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors-intracellular lipid droplets-and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  20. Lacrimal Gland Inflammation Deregulates Extracellular Matrix Remodeling and Alters Molecular Signature of Epithelial Stem/Progenitor Cells

    PubMed Central

    Umazume, Takeshi; Thomas, William M.; Campbell, Sabrina; Aluri, Hema; Thotakura, Suharika; Zoukhri, Driss; Makarenkova, Helen P.

    2015-01-01

    Purpose The adult lacrimal gland (LG) is highly regenerative and is able to repair itself even after substantial damage; however, this ability to regenerate is lost with the development of dry eye conditions in chronically inflamed LGs.This study compares changes in the cell adhesion and cell matrix molecules and stem cell transcription factors in the LGs of healthy mice and of two mouse models of Sjögren's syndrome: nonobese diabetic (NOD) and MRL-lpr/lpr (MRL/lpr) mice during the early stage of inflammation. Methods The LGs from 12- to 13-week-old female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for quantitative (q) RT-PCR and qRT-PCR Arrays, histology, immunohistochemistry, and Western blotting. Results The extracellular matrix (ECM) and adhesion molecules RT2-PCR array combined with protein expression data revealed changes in the expression of integrins, matrix metalloproteinases, and other molecules, which are associated largely with invasion, attachment, and expansion of the lymphocytic cells, whereas changes in the stem cell transcription factors revealed substantial decrease in expression of transcription factors associated with epithelial stem/progenitor cell lineage. Conclusions We concluded that the expression of several important ECM components is significantly deregulated in the LG of two murine models of Sjögren's syndrome, suggesting an alteration of the epithelial stem/progenitor cell niche. This may result in profound effects on localization, activation, proliferation, and differentiation of the LG stem/progenitor cells and, therefore, LG regeneration. PMID:26747770

  1. Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments

    PubMed Central

    Flitney, Eric W.; Kuczmarski, Edward R.; Adam, Stephen A.; Goldman, Robert D.

    2009-01-01

    The effects of shear stress on the keratin intermediate filament (KIF) cytoskeleton of cultured human alveolar epithelial (A549) cells have been investigated. Under normal culture conditions, immunofluorescence revealed a delicate network of fine tonofibrils containing KIFs, together with many nonfilamentous, keratin-containing “particles,” mostly containing either keratin 8 (K8) or 18 (K18), but not both. Triton X-100 extracted ∼10% of the cellular keratin, and this was accompanied by a loss of the particles but not the KIFs. Shear stress dramatically reduced the soluble keratin component and transformed the fine bundles of KIFs into thicker, “wavy” tonofibrils. Both effects were accompanied by the disappearance of most keratin particles and by increased phosphorylation of K8 and K18 on serine residues 73 and 33, respectively. The particles that remained after shearing were phosphorylated and were closely associated with KIFs. We suggest that keratin particles constitute a reservoir of protein that can be recruited into KIFs under flow, creating a more robust cytoskeleton able to withstand shear forces more effectively.—Flitney, E. W., Kuczmarski, E. R., Adam, S. A., Goldman, R. D. Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments. PMID:19246484

  2. EB1-recruited microtubule +TIP complexes coordinate protrusion dynamics during 3D epithelial remodeling

    PubMed Central

    Gierke, Sarah; Wittmann, Torsten

    2012-01-01

    SUMMARY Background Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays a central role in development and disease of multicellular organisms. Although dynamic microtubules (MTs) are required for directed migration on flat surfaces, how MT dynamics are controlled or contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We use confocal live cell imaging to analyze MT function and dynamics during 3D epithelial morphogenesis and remodeling of polarized Madin-Darby canine kidney (MDCK) epithelial cells that undergo partial epithelial-to-mesenchymal transition (EMT) in response to hepatocyte growth factor (HGF). Results We find that HGF treatment increases MT growth rate before morphological changes are evident, and that large numbers of MTs grow into HGF-induced cell extensions independent of centrosome reorientation. Using lentivirus-mediated shRNA, we demonstrate that EB1, an adaptor protein that mediates recruitment of numerous other +TIP proteins to growing MT plus ends, is required for this HGF-induced MT reorganization. We further show that protrusion and adhesion dynamics are disorganized, and that vesicular trafficking to the tip of HGF-induced cell extensions is disrupted in EB1-depleted cells. Conclusions We conclude that EB1-mediated interactions with growing MTs are important to coordinate cell shape changes and directed migration into the surrounding extracellular matrix during epithelial remodeling in a physiological 3D environment. In contrast, EB1 is not required for the establishment or maintenance of apical-basal cell polarity, suggesting different functions of +TIPs and MTs in different types of cell polarity. PMID:22483942

  3. Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

    PubMed Central

    Lapperre, Thérèse S; Sont, Jacob K; van Schadewijk, Annemarie; Gosman, Margot ME; Postma, Dirkje S; Bajema, Ingeborg M; Timens, Wim; Mauad, Thais; Hiemstra, Pieter S

    2007-01-01

    Background Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation. Methods 114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV1 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue+) area (%), proliferating (Ki-67+) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies. Results Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001). Conclusion Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years). Trial registration NCT00158847 PMID:18039368

  4. Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

    PubMed Central

    Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

    2016-01-01

    Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

  5. SPRY1 regulates mammary epithelial morphogenesis by modulating EGFR-dependent stromal paracrine signaling and ECM remodeling.

    PubMed

    Koledova, Zuzana; Zhang, Xiaohong; Streuli, Charles; Clarke, Robert B; Klein, Ophir D; Werb, Zena; Lu, Pengfei

    2016-09-27

    The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development. PMID:27621461

  6. Inhalation exposure to ethylene induces eosinophilic rhinitis and nasal epithelial remodeling in Fischer 344 rats.

    PubMed

    Brandenberger, Christina; Hotchkiss, Jon A; Krieger, Shannon M; Pottenger, Lynn H; Harkema, Jack R

    2015-11-01

    This study investigated the time- and concentration-dependent effects of inhaled ethylene on eosinophilic rhinitis and nasal epithelial remodeling in Fisher 344 rats exposed to 0, 10, 50, 300, or 10,000 ppm ethylene, 6 h/day, 5 days/week for up to 4 weeks. Morphometric quantitation of eosinophilic inflammation and mucous cell metaplasia/hyperplasia (MCM) and nasal mucosal gene expression were evaluated at anatomic sites previously shown to undergo ethylene-induced epithelial remodeling. Serum levels of total IgE, IgG1 and IgG2a were measured to determine if ethylene exposure increased the expression of Th2-associated (IgE and IgG1) relative to Th1-associated (IgG2a) antibody isotypes. Rats exposed to 0 or 10,000 ppm for 1, 3, 5, 10, or 20 days were analyzed to assess the temporal pattern of ethylene-induced alterations in nasal epithelial cell proliferation, morphology and gene expression. Rats exposed to 0, 10, 50, 300, and 10,000 ppm ethylene for 20 days were analyzed to assess concentration-dependent effects on lesion development. Additional rats exposed 4 weeks to 0, 300, or 10,000 ppm ethylene were held for 13 weeks post-exposure to examine the persistence of ethylene-induced mucosal alterations. The data indicate that cell death and reparative cell proliferation were not a part of the pathogenesis of ethylene-induced nasal lesions. Enhanced gene expression of Th2 cytokines (e.g., IL-5, IL-13) and chitinase (YM1/2) in the nasal mucosa was much greater than that of Th1 cytokines (e.g., IFNγ) after ethylene exposure. A significant increase in MCM was measured after 5 days of exposure to 10,000 ppm ethylene and after 20 days of exposure 10 ppm ethylene. Ethylene-induced MCM was reversible after cessation of exposure. No increase in total serum IgE, IgG1 or IgG2a was measured in any ethylene-exposed group. These data do not support involvement of an immune-mediated allergic mechanism in the pathogenesis of ethylene-induced nasal lesions in rats. Repeated

  7. Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.

    PubMed

    Le Cras, Timothy D; Acciani, Thomas H; Mushaben, Elizabeth M; Kramer, Elizabeth L; Pastura, Patricia A; Hardie, William D; Korfhagen, Thomas R; Sivaprasad, Umasundari; Ericksen, Mark; Gibson, Aaron M; Holtzman, Michael J; Whitsett, Jeffrey A; Hershey, Gurjit K Khurana

    2011-03-01

    Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.

  8. 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.

  9. Corneal Epithelial Remodeling and Its Effect on Corneal Asphericity after Transepithelial Photorefractive Keratectomy for Myopia.

    PubMed

    Hou, Jie; Wang, Yan; Lei, Yulin; Zheng, Xiuyun; Zhang, Ying

    2016-01-01

    Purpose. To evaluate the changes in epithelial thickness profile following transepithelial photorefractive keratectomy (T-PRK) for myopia and to investigate the effect of epithelial remodeling on corneal asphericity. Methods. Forty-four patients (44 right eyes) who underwent T-PRK were retrospectively evaluated. Epithelial thickness was measured using spectral-domain optical coherence tomography at different corneal zones (central, 2 mm; paracentral, 2-5 mm; and mid-peripheral, 5-6 mm) preoperatively and at 1 week and 1, 3, and 6 months postoperatively. The correlation between the changes in corneal epithelial thickness (ΔCET) and postoperative Q-value changes (ΔQ) was analyzed 6 months postoperatively. Results. Epithelial thickness at 6 months showed a negative meniscus-like lenticular pattern with less central thickening, which increased progressively toward the mid-periphery (3.69 ± 4.2, 5.19 ± 3.8, and 6.23 ± 3.9 μm at the center, paracenter, and mid-periphery, resp., P < 0.01). A significant positive relationship was observed between epithelial thickening and ΔQ 6 months postoperatively (r = 0.438, 0.580, and 0.504, resp., P < 0.01). Conclusions. Significant epithelial thickening was observed after T-PRK and showed a lenticular change with more thickening mid-peripherally, resulting in increased oblateness postoperatively. Epithelial remodeling may modify the epithelial thickness profile after surface ablation refractive surgery for myopia. PMID:27672447

  10. Corneal Epithelial Remodeling and Its Effect on Corneal Asphericity after Transepithelial Photorefractive Keratectomy for Myopia

    PubMed Central

    Zheng, Xiuyun

    2016-01-01

    Purpose. To evaluate the changes in epithelial thickness profile following transepithelial photorefractive keratectomy (T-PRK) for myopia and to investigate the effect of epithelial remodeling on corneal asphericity. Methods. Forty-four patients (44 right eyes) who underwent T-PRK were retrospectively evaluated. Epithelial thickness was measured using spectral-domain optical coherence tomography at different corneal zones (central, 2 mm; paracentral, 2–5 mm; and mid-peripheral, 5-6 mm) preoperatively and at 1 week and 1, 3, and 6 months postoperatively. The correlation between the changes in corneal epithelial thickness (ΔCET) and postoperative Q-value changes (ΔQ) was analyzed 6 months postoperatively. Results. Epithelial thickness at 6 months showed a negative meniscus-like lenticular pattern with less central thickening, which increased progressively toward the mid-periphery (3.69 ± 4.2, 5.19 ± 3.8, and 6.23 ± 3.9 μm at the center, paracenter, and mid-periphery, resp., P < 0.01). A significant positive relationship was observed between epithelial thickening and ΔQ 6 months postoperatively (r = 0.438, 0.580, and 0.504, resp., P < 0.01). Conclusions. Significant epithelial thickening was observed after T-PRK and showed a lenticular change with more thickening mid-peripherally, resulting in increased oblateness postoperatively. Epithelial remodeling may modify the epithelial thickness profile after surface ablation refractive surgery for myopia.

  11. Corneal Epithelial Remodeling and Its Effect on Corneal Asphericity after Transepithelial Photorefractive Keratectomy for Myopia

    PubMed Central

    Zheng, Xiuyun

    2016-01-01

    Purpose. To evaluate the changes in epithelial thickness profile following transepithelial photorefractive keratectomy (T-PRK) for myopia and to investigate the effect of epithelial remodeling on corneal asphericity. Methods. Forty-four patients (44 right eyes) who underwent T-PRK were retrospectively evaluated. Epithelial thickness was measured using spectral-domain optical coherence tomography at different corneal zones (central, 2 mm; paracentral, 2–5 mm; and mid-peripheral, 5-6 mm) preoperatively and at 1 week and 1, 3, and 6 months postoperatively. The correlation between the changes in corneal epithelial thickness (ΔCET) and postoperative Q-value changes (ΔQ) was analyzed 6 months postoperatively. Results. Epithelial thickness at 6 months showed a negative meniscus-like lenticular pattern with less central thickening, which increased progressively toward the mid-periphery (3.69 ± 4.2, 5.19 ± 3.8, and 6.23 ± 3.9 μm at the center, paracenter, and mid-periphery, resp., P < 0.01). A significant positive relationship was observed between epithelial thickening and ΔQ 6 months postoperatively (r = 0.438, 0.580, and 0.504, resp., P < 0.01). Conclusions. Significant epithelial thickening was observed after T-PRK and showed a lenticular change with more thickening mid-peripherally, resulting in increased oblateness postoperatively. Epithelial remodeling may modify the epithelial thickness profile after surface ablation refractive surgery for myopia. PMID:27672447

  12. 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

  13. Retinal pigment epithelial cell proliferation

    PubMed Central

    Temple, Sally

    2015-01-01

    The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most in vitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration. PMID:26041390

  14. Eosinophilic Esophagitis: Epithelial Mesenchymal Transition Contributes to Esophageal Remodeling and Reverses with Treatment

    PubMed Central

    Kagalwalla, Amir F.; Akhtar, Noorain; Woodruff, Samantha A.; Rea, Bryan A.; Masterson, Joanne C.; Mukkada, Vincent; Parashette, Kalyan R.; Du, Jian; Fillon, Sophie; Protheroe, Cheryl A.; Lee, James J.; Amsden, Katie; Melin-Aldana, Hector; Capocelli, Kelley E.; Furuta, Glenn T.; Ackerman, Steven J.

    2012-01-01

    Background Mechanisms underlying esophageal remodeling with subepithelial fibrosis in eosinophilic esophagitis (EoE) have not been delineated. Objectives To explore a role for Epithelial Mesenchymal Transition (EMT) in EoE, and whether EMT resolves with treatment. Methods Esophageal biopsies from 60 children were immunostained for epithelial (cytokeratin) and mesenchymal (vimentin) EMT biomarkers, and EMT quantified. Subjects studied had EoE (n=17), EoE-indeterminate (n=15), GERD (n=7) or normal esophagus (n=21). EMT was analyzed for relationships to diagnosis, eosinophils, and indices of subepithelial fibrosis, eosinophil peroxidase (EPX) and TGF-β immunostaining. EMT was assessed in pre- and post-treatment biopsies from 18 EoE subjects treated with elemental diet, six-food elimination diet, or topical corticosteroids (n=6/group). Results TGF-β1 treatment of esophageal epithelial cells in vitro for 24hrs induced upregulation of mesenchymal genes characteristic of EMT including N-cadherin (3.3-fold), vimentin (2.1-fold) and fibronectin (7.5-fold). EMT in esophageal biopsies was associated with EoE (or indeterminate EoE), but not GERD or normal esophagus, and was correlated to eosinophils (r=0.691), EPX (r=0.738) and TGF-β (r=0.520) immunostaining, and fibrosis (r=0.644) indices. EMT resolved with EoE treatments that induced clinicopathologic remission with reduced eosinophils. EMT decreased significantly post-treatment by 74.1% overall in the 18 treated EoE subjects; pre- vs. post-treatment EMT scores–3.17±0.82 vs. 0.82±0.39 (p<0.001), with similar decreases within treatment groups. Pre-/post-treatment EMT was strongly correlated with eosinophils for combined (r=0.804, p< 0.001) and individual treatment groups. Conclusions EMT likely contributes to subepithelial fibrosis in EoE, resolves with treatments that decrease esophageal inflammation, and its resolution correlates with decreased numbers of esophageal eosinophils. PMID:22465212

  15. 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

  16. 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.

  17. 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.

  18. Progenitor cells in pulmonary vascular remodeling.

    PubMed

    Yeager, Michael E; Frid, Maria G; Stenmark, Kurt R

    2011-01-01

    Pulmonary hypertension is characterized by cellular and structural changes in the walls of pulmonary arteries. Intimal thickening and fibrosis, medial hypertrophy and fibroproliferative changes in the adventitia are commonly observed, as is the extension of smooth muscle into the previously non-muscularized vessels. A majority of these changes are associated with the enhanced presence of α-SM-actin+ cells and inflammatory cells. Atypical abundances of functionally distinct endothelial cells, particularly in the intima (plexiform lesions), and also in the perivascular regions, are also described. At present, neither the origin(s) of these cells nor the molecular mechanisms responsible for their accumulation, in any of the three compartments of the vessel wall, have been fully elucidated. The possibility that they arise from either resident vascular progenitors or bone marrow-derived progenitor cells is now well established. Resident vascular progenitor cells have been demonstrated to exist within the vessel wall, and in response to certain stimuli, to expand and express myofibroblastic, endothelial or even hematopoietic markers. Bone marrow-derived or circulating progenitor cells have also been shown to be recruited to sites of vascular injury and to assume both endothelial and SM-like phenotypes. Here, we review the data supporting the contributory role of vascular progenitors (including endothelial progenitor cells, smooth muscle progenitor cells, pericytes, and fibrocytes) in vascular remodeling. A more complete understanding of the processes by which progenitor cells modulate pulmonary vascular remodeling will undoubtedly herald a renaissance of therapies extending beyond the control of vascular tonicity and reduction of pulmonary artery pressure. PMID:22034593

  19. Cell wall remodeling under abiotic stress

    PubMed Central

    Tenhaken, Raimund

    2015-01-01

    Plants exposed to abiotic stress respond to unfavorable conditions on multiple levels. One challenge under drought stress is to reduce shoot growth while maintaining root growth, a process requiring differential cell wall synthesis and remodeling. Key players in this process are the formation of reactive oxygen species (ROS) and peroxidases, which initially cross-link phenolic compounds and glycoproteins of the cell walls causing stiffening. The function of ROS shifts after having converted all the peroxidase substrates in the cell wall. If ROS-levels remain high during prolonged stress, OH°-radicals are formed which lead to polymer cleavage. In concert with xyloglucan modifying enzymes and expansins, the resulting cell wall loosening allows further growth of stressed organs. PMID:25709610

  20. mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells

    PubMed Central

    2014-01-01

    Background Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. Methods Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. Results Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. Conclusions Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs. PMID:24678619

  1. 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

  2. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  3. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells.

    PubMed

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na(+) channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  4. CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

    PubMed Central

    Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

    2016-01-01

    Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate

  5. Dpp signaling directs cell motility and invasiveness during epithelial morphogenesis.

    PubMed

    Ninov, Nikolay; Menezes-Cabral, Sofia; Prat-Rojo, Carla; Manjón, Cristina; Weiss, Alexander; Pyrowolakis, George; Affolter, Markus; Martín-Blanco, Enrique

    2010-03-23

    Tissue remodeling in development and disease involves the coordinated invasion of neighboring territories and/or the replacement of entire cell populations. Cell guidance, cell matching, transitions from passive to migratory epithelia, cell growth and death, and extracellular matrix remodeling all impinge on epithelial spreading. Significantly, the extracellular signals that direct these activities and the specific cellular elements and mechanisms regulated by these signals remain in most cases to be identified. To address these issues, we performed an analysis of histoblasts (Drosophila abdominal epithelial founder cells) on their transition from a dormant state to active migration replacing obsolete larval epidermal cells (LECs). We found that during expansion, Decapentaplegic (Dpp) secreted from surrounding LECs leads to graded pathway activation in cells at the periphery of histoblast nests. Across nests, Dpp activity confers differential cellular behavior and motility by modulating cell-cell contacts, the organization and activity of the cytoskeleton, and histoblast attachment to the substrate. Furthermore, Dpp also prevents the premature death of LECs, allowing the coordination of histoblast expansion to LEC delamination. Dpp signaling activity directing histoblast spreading and invasiveness mimics transforming growth factor-beta and bone morphogenetic proteins' role in enhancing the motility and invasiveness of cancer cells, resulting in the promotion of metastasis. PMID:20226662

  6. Lymphoid Tissue Mesenchymal Stromal Cells in Development and Tissue Remodeling

    PubMed Central

    2016-01-01

    Secondary lymphoid organs (SLOs) are sites that facilitate cell-cell interactions required for generating adaptive immune responses. Nonhematopoietic mesenchymal stromal cells have been shown to play a critical role in SLO function, organization, and tissue homeostasis. The stromal microenvironment undergoes profound remodeling to support immune responses. However, chronic inflammatory conditions can promote uncontrolled stromal cell activation and aberrant tissue remodeling including fibrosis, thus leading to tissue damage. Despite recent advancements, the origin and role of mesenchymal stromal cells involved in SLO development and remodeling remain unclear. PMID:27190524

  7. Remodeling of Cell-Cell Junctions in Arrhythmogenic Cardiomyopathy

    PubMed Central

    Asimaki, Angeliki; Saffitz, Jeffrey E.

    2015-01-01

    Arrhythmogenic cardiomyopathy (AC) is a primary myocardial disorder characterized by a high incidence of ventricular arrhythmias often preceding the onset of ventricular remodeling and dysfunction. Approximately 50% of patients diagnosed with AC have one or more mutations in genes encoding desmosomal proteins, although non-desmosomal genes have also been associated with the disease. Increasing evidence implicates remodeling of intercalated disk proteins reflecting abnormal responses to mechanical load and aberrant cell signaling pathways in the pathogenesis of AC. This review summarizes recent advances in understanding disease mechanisms in AC that have come from studies of human myocardium and experimental models. PMID:24460198

  8. Secretory component: a glandular epithelial cell marker.

    PubMed Central

    Harris, J. P.; South, M. A.

    1981-01-01

    Secretory component (SC) has been demonstrated to be produced by both normal and malignantly transformed glandular epithelial cells. By an indirect immunofluorescent technique, this study surveys tumors of varied cellular origin in order to determine the reliability of SC as a marker for tumor cells derived from glandular epithelium. Both primary and metastatic tumors of glandular epithelial origin demonstrated SC fluorescence, while nonglandular epithelial tumors did not. This observation was extended to live single-cell preparations, which demonstrated intense cell-surface fluorescence only when glandular epithelial tumors cells were examined. Additionally, fixed, cytocentrifuged, single-cell preparations of glandular epithelial tumors demonstrated cytoplasmic SC fluorescence. When breast carcinoma was examined, all cases demonstrated SC, regardless of the degree of differentiation. This assay appears to have useful clinical application in that the finding of SC provides indication of the glandular epithelial origin of a malignantly transformed cell. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6271014

  9. Hyperoxia alters the mechanical properties of alveolar epithelial cells.

    PubMed

    Roan, Esra; Wilhelm, Kristina; Bada, Alex; Makena, Patrudu S; Gorantla, Vijay K; Sinclair, Scott E; Waters, Christopher M

    2012-06-15

    Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability. PMID:22467640

  10. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

  11. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    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. PMID:26656655

  12. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis.

    PubMed

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    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. PMID:26656655

  13. LOXL2 in epithelial cell plasticity and tumor progression.

    PubMed

    Cano, Amparo; Santamaría, Patricia G; Moreno-Bueno, Gema

    2012-09-01

    Several members of the lysyl oxidase family have recently emerged as important regulators of tumor progression. Among them, LOXL2 has been shown to be involved in tumor progression and metastasis of several tumor types, including breast carcinomas. Secreted LOXL2 participates in the remodeling of the extracellular matrix of the tumor microenvironment, in a similar fashion to prototypical lysyl oxidase. In addition, new intracellular functions of LOXL2 have been described, such as its involvement in the regulation of the epithelial-to-mesenchymal transition, epithelial cell polarity and differentiation mediated by transcriptional repression mechanisms. Importantly, intracellular (perinuclear) expression of LOXL2 is associated with poor prognosis and distant metastasis of specific tumor types, such as larynx squamous cell carcinoma and basal breast carcinomas. These recent findings open new avenues for the therapeutic utility of LOXL2.

  14. Clara epithelial cell depletion in the lung.

    PubMed

    Sonar, Sanchaita S; Dudda, Jan C

    2013-01-01

    The bronchial epithelium has been increasingly recognized as an important immunomodulatory compartment in asthma and other lung diseases. Clara cells, which comprise the nonciliated secretory epithelial cells, are an important epithelial cell type with functions in the regulation of lung homeostasis and inflammation. Using naphthalene, Clara cells can be depleted within 24 h and regenerate by 1 month, hence, providing an easy method to study the impact of Clara cells on lung inflammation.

  15. Human glomerular epithelial cell proteoglycans

    SciTech Connect

    Thomas, G.J.; Jenner, L.; Mason, R.M.; Davies, M. )

    1990-04-01

    Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate.

  16. Control of cell mechanics by RhoA and calcium fluxes during epithelial scattering.

    PubMed

    Haws, Hillary J; McNeil, Melissa A; Hansen, Marc D H

    2016-01-01

    Epithelial tissues use adherens junctions to maintain tight interactions and coordinate cellular activities. Adherens junctions are remodeled during epithelial morphogenesis, including instances of epithelial-mesenchymal transition, or EMT, wherein individual cells detach from the tissue and migrate as individual cells. EMT has been recapitulated by growth factor induction of epithelial scattering in cell culture. In culture systems, cells undergo a highly reproducible series of cell morphology changes, most notably cell spreading followed by cellular compaction and cell migration. These morphology changes are accompanied by striking actin rearrangements. The current evidence suggests that global changes in actomyosin-based cellular contractility, first a loss of contractility during spreading and its activation during cell compaction, are the main drivers of epithelial scattering. In this review, we focus on how spreading and contractility might be controlled during epithelial scattering. While we propose a central role for RhoA, which is well known to control cellular contractility in multiple systems and whose role in epithelial scattering is well accepted, we suggest potential roles for additional cellular systems whose role in epithelial cell biology has been less well documented. In particular, we propose critical roles for vesicle recycling, calcium channels, and calcium-dependent kinases. PMID:27583192

  17. Remodeling of Endogenous Mammary Epithelium by Breast Cancer Stem Cells

    PubMed Central

    Parashurama, Natesh; Lobo, Neethan A.; Ito, Ken; Mosley, Adriane R.; Habte, Frezghi G.; Zabala, Maider; Smith, Bryan R.; Lam, Jessica; Weissman, Irving L.; Clarke, Michael F.; Gambhir, Sanjiv S.

    2014-01-01

    Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC. PMID:22899386

  18. Remodeling of endogenous mammary epithelium by breast cancer stem cells.

    PubMed

    Parashurama, Natesh; Lobo, Neethan A; Ito, Ken; Mosley, Adriane R; Habte, Frezghi G; Zabala, Maider; Smith, Bryan R; Lam, Jessica; Weissman, Irving L; Clarke, Michael F; Gambhir, Sanjiv S

    2012-10-01

    Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC. PMID:22899386

  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. Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.

    PubMed

    Hynds, Robert E; Giangreco, Adam

    2013-03-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. PMID:23203919

  1. Campylobacter concisus pathotypes induce distinct global responses in intestinal epithelial cells

    PubMed Central

    Deshpande, Nandan P.; Wilkins, Marc R.; Castaño-Rodríguez, Natalia; Bainbridge, Emily; Sodhi, Nidhi; Riordan, Stephen M.; Mitchell, Hazel M.; Kaakoush, Nadeem O.

    2016-01-01

    The epithelial response to the opportunistic pathogen Campylobacter concisus is poorly characterised. Here, we assessed the intestinal epithelial responses to two C. concisus strains with different virulence characteristics in Caco-2 cells using RNAseq, and validated a subset of the response using qPCR arrays. C. concisus strains induced distinct response patterns from intestinal epithelial cells, with the toxigenic strain inducing a significantly more amplified response. A range of cellular functions were significantly regulated in a strain-specific manner, including epithelial-to-mesenchymal transition (NOTCH and Hedgehog), cytoskeletal remodeling, tight junctions, inflammatory responses and autophagy. Pattern recognition receptors were regulated, including TLR3 and IFI16, suggesting that nucleic acid sensing was important for epithelial recognition of C. concisus. C. concisus zonula occludens toxin (ZOT) was expressed and purified, and the epithelial response to the toxin was analysed using RNAseq. ZOT upregulated PAR2 expression, as well as processes related to tight junctions and cytoskeletal remodeling. C. concisus ZOT also induced upregulation of TLR3, pro-inflammatory cytokines IL6, IL8 and chemokine CXCL16, as well as the executioner caspase CASP7. Here, we characterise distinct global epithelial responses to C. concisus strains, and the virulence factor ZOT, and provide novel information on mechanisms by which this bacterium may affect the host. PMID:27677841

  2. ATP Dependent Chromatin Remodeling Enzymes in Embryonic Stem Cells

    PubMed Central

    Saladi, Srinivas Vinod

    2010-01-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure. PMID:20148317

  3. Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

    PubMed

    Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena; Gabrielsson, Susanne; Rådmark, Olof

    2016-09-01

    Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung. PMID:27436590

  4. The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation.

    PubMed

    Wright, Bernice; Hopkinson, Andrew; Leyland, Martin; Connon, Che J

    2013-01-01

    Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P≤0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

  5. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  6. Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties.

    PubMed

    Tsunematsu, Takaaki; Fujiwara, Natsumi; Yoshida, Maki; Takayama, Yukihiro; Kujiraoka, Satoko; Qi, Guangying; Kitagawa, Masae; Kondo, Tomoyuki; Yamada, Akiko; Arakaki, Rieko; Miyauchi, Mutsumi; Ogawa, Ikuko; Abiko, Yoshihiro; Nikawa, Hiroki; Murakami, Shinya; Takata, Takashi; Ishimaru, Naozumi; Kudo, Yasusei

    2016-10-01

    Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of the Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. ERM cells are unique epithelial cells that remain in periodontal tissues throughout adult life. They have a functional role in the repair/regeneration of cement or enamel. Here, we isolated odontogenic epithelial cells from ERM in the periodontal ligament, and the cells were spontaneously immortalized. Immortalized odontogenic epithelial (iOdE) cells had the ability to form spheroids and expressed stem cell-related genes. Interestingly, iOdE cells underwent osteogenic differentiation, as demonstrated by the mineralization activity in vitro in mineralization-inducing media and formation of calcification foci in iOdE cells transplanted into immunocompromised mice. These findings suggest that a cell population with features similar to stem cells exists in ERM and that this cell population has a differentiation capacity for producing calcifications in a particular microenvironment. In summary, iOdE cells will provide a convenient cell source for tissue engineering and experimental models to investigate tooth growth, differentiation, and tumorigenesis. PMID:27479086

  7. Wound healing of intestinal epithelial cells

    PubMed Central

    Iizuka, Masahiro; Konno, Shiho

    2011-01-01

    The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing. PMID:21633524

  8. Physical principles of membrane remodelling during cell mechanoadaptation

    PubMed Central

    Kosmalska, Anita Joanna; Casares, Laura; Elosegui-Artola, Alberto; Thottacherry, Joseph Jose; Moreno-Vicente, Roberto; González-Tarragó, Víctor; del Pozo, Miguel Ángel; Mayor, Satyajit; Arroyo, Marino; Navajas, Daniel; Trepat, Xavier; Gauthier, Nils C.; Roca-Cusachs, Pere

    2015-01-01

    Biological processes in any physiological environment involve changes in cell shape, which must be accommodated by their physical envelope—the bilayer membrane. However, the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear. Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process. This process is passive, local, almost instantaneous, before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane. We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell–substrate interface. Once formed, cells reabsorb the invaginations through an active process with duration of the order of minutes. PMID:26073653

  9. Listeria monocytogenes Invades the Epithelial Junctions at Sites of Cell Extrusion

    PubMed Central

    Pentecost, Mickey; Otto, Glen

    2006-01-01

    Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier. PMID:16446782

  10. 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...

  11. Replication of cultured lung epithelial cells

    SciTech Connect

    Guzowski, D.; Bienkowski, R.

    1986-03-05

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

  12. Cell-surface remodelling during mammalian erythropoiesis.

    PubMed

    Wraith, D C; Chesterton, C J

    1982-10-15

    Current evidence suggests that the major cell-surface modification occurring during mammalian erythropoiesis could be generated by two separate mechanisms: either selective loss of membrane proteins during enucleation or endocytosis at the subsequent reticulocyte and erythrocyte stages. The former idea was tested by collecting developing rabbit erythroid cells before and after the enucleation step and comparing their cell-surface protein composition via radiolabelling and electrophoresis. Few changes were observed. Our data thus lend support to the endocytosis mechanism.

  13. Control of the epithelial stem cell epigenome: the shaping of epithelial stem cell identity.

    PubMed

    Iglesias-Bartolome, Ramiro; Callejas-Valera, Juan Luis; Gutkind, J Silvio

    2013-04-01

    The squamous epithelium covering the skin and oral mucosa relies on epithelial stem cells for tissue renewal. Dynamic changes in DNA methylation, histone methylation and acetylation, and higher order chromatin structure are required to preserve their self-renewal capacity while orchestrating the timely execution of cell differentiation programs. This complex network of epigenetic modifications shapes the epithelial stem cell identity and fate. Pathological alterations can be perceived by aberrant chromatin sensors, such as the INK4/ARF locus, which initiate tumor suppressive cell senescence programs, and can often result in epithelial stem cell exhaustion. Unveiling the mechanisms controlling the epigenome in epithelial stem cells may help protect against the loss of their tissue regenerative capacity, thereby preventing premature aging without increasing cancer risk. PMID:23434069

  14. Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates.

    PubMed

    Kawamura, Kazuo; Sugino, Yasuo; Sunanaga, Takeshi; Fujiwara, Shigeki

    2008-01-01

    The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.

  15. Human tracheobronchial basal cells. Normal versus remodeling/repairing phenotypes in vivo and in vitro.

    PubMed

    Ghosh, Moumita; Ahmad, Shama; Jian, Abhilasha; Li, Bilan; Smith, Russell W; Helm, Karen M; Seibold, Max A; Groshong, Steven D; White, Carl W; Reynolds, Susan D

    2013-12-01

    Human tracheobronchial epithelial (TBE) basal cells (BCs) function as progenitors in normal tissue. However, mechanistic studies are typically performed in vitro and frequently use BCs recovered from patients who die of nonrespiratory disease. It is not known whether the cadaveric epithelium (1) is undergoing homeostatic remodeling and/or repair, or (2) yields BC clones that represent homeostatic processes identified in tissue. We sought to compare the phenotype of TBE-BCs with that of BCs cultured under optimal clone-forming conditions. TBE pathology was evaluated using quantitative histomorphometry. The cultured BC phenotype was determined by fluorescence-activated cell sorter analysis. Clone organization and cell phenotype were determined by immunostaining. The cadaveric TBE is 20% normal. In these regions, BCs are keratin (K)-5(+) and tetraspanin CD151(+), and demonstrate a low mitotic index. In contrast, 80% of the cadaveric TBE exhibits homeostatic remodeling/repair processes. In these regions, BCs are K5(+)/K14(+), and a subset expresses tissue factor (TF). Passage 1 TBE cells are BCs that are K5(+)/TF(+), and half coexpress CD151. Optimal clone formation conditions use an irradiated NIH3T3 fibroblast feeder layer (American Type Culture Collection, Frederick, MD) and serum-supplemented Epicult-B medium (Stemcell Technologies, La Jolla, CA). The TF(+)/CD151(-) BC subpopulation is the most clonogenic BC subtype, and is enriched with K14(+) cells. TF(+)/CD151(-) BCs generate clones containing BCs that are K5(+)/Trp63(+), but K14(-)/CD151(-). TF(+) cells are limited to the clone edge. In conclusion, clonogenic human TBE BCs (1) exhibit a molecular phenotype that is a composite of the normal and remodeling/reparative BC phenotypes observed in tissue, and (2) generate organoid clones that contain phenotypically distinct BC subpopulations.

  16. 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.

  17. 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

  18. Expression of cyclin D{sub 1} during endotoxin-induced aleveolar type II cell hyperplasia in rat lung and the detection of apoptotic cells during the remodeling process

    SciTech Connect

    Tesfaigzi, J.; Wood, M.B.; Johnson, N.F.

    1995-12-01

    Our studies have shown that endotoxin intratracheally instilled into the rat lung induces proliferation of alveolar type II cells. In that study, the alveolar type II cells. In that study, the alveolar type II cell hyperplasia occurred 2 d after instillation of endotoxin and persisted for a further 2 d. After hyperplasia, the lung remodeled and returned to a normal state within 24-48 h. Understanding the mechanisms involved in the remodeling process of this transient hyperplasia may be useful to identify molecular changes that are altered in neoplasia. The purpose of the present study was to corroborate induction of epithelial cell hyperplasia by endotoxin and to delineate mechanisms involved in tissue remodeling after endotoxin-induced alveolar type II cell hyperplasia. In conclusion, immonostaining with cyclin D1 and cytokeratin shows that endotoxin induced epithelial cell proliferation and resulted in hyperplasia in the lung which persisted through 4 d post-instillation.

  19. Human bronchial epithelial cells express and secrete MMP-12.

    PubMed

    Lavigne, Mark C; Thakker, Paresh; Gunn, Jason; Wong, Anthony; Miyashiro, Joy S; Wasserman, Aeona M; Wei, Shui-Qing; Pelker, Jeffrey W; Kobayashi, Michiko; Eppihimer, Michael J

    2004-11-12

    Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, which may be responsible for enlargement of alveoli in chronic obstructive pulmonary disease (COPD) and remodeling of pulmonary tissue associated with chronic asthma. Here, we provide novel evidence that MMP-12 is expressed and secreted by normal human bronchial epithelial cell cultures (NHBECs) and reveal the regulation of MMP-12 gene expression by tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF), and interferon gamma (IFN-gamma). Reverse transcription-polymerase chain reaction analyses demonstrated MMP-12 mRNA presence in unstimulated differentiated NHBEC cultures. Cultures stimulated independently with EGF or IFN-gamma failed to alter MMP-12 mRNA abundance, while TNF-alpha, TNF-alpha+EGF, or TNF-alpha+IFN-gamma elicited relatively early (6 h) peak increases in MMP-12 mRNA levels. Western blot analyses specifically indicated the presence of MMP-12 in differentiated NHBEC-conditioned media. These findings indicate that the bronchial epithelium may be an important source of elastolytic activity in COPD and tissue remodeling in chronic asthma.

  20. Respiratory epithelial cells orchestrate pulmonary innate immunity

    PubMed Central

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and ‘instruct’ the professional immune system to protect the lungs from infection and injury. PMID:25521682

  1. Cell Wall Composition, Biosynthesis and Remodeling during Pollen Tube Growth

    PubMed Central

    Mollet, Jean-Claude; Leroux, Christelle; Dardelle, Flavien; Lehner, Arnaud

    2013-01-01

    The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed. PMID:27137369

  2. Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

    PubMed

    Gavara, Núria; Sunyer, Raimon; Roca-Cusachs, Pere; Farré, Ramon; Rotger, Mar; Navajas, Daniel

    2006-08-01

    Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung. PMID:16675616

  3. Ionizing radiation induces heritable disruption of epithelial cell interactions

    NASA Technical Reports Server (NTRS)

    Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Chatterjee, A. (Principal Investigator)

    2003-01-01

    Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

  4. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

  5. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

    PubMed Central

    Boraas, Liana C.; Guidry, Julia B.; Pineda, Emma T.; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this

  6. Epithelial expression of profibrotic mediators in a model of allergen-induced airway remodeling.

    PubMed

    Kelly, Margaret M; Leigh, Richard; Bonniaud, Philippe; Ellis, Russ; Wattie, Jennifer; Smith, Mary Jo; Martin, Gail; Panju, Mohammed; Inman, Mark D; Gauldie, Jack

    2005-02-01

    Airway remodeling, including subepithelial fibrosis, is a characteristic feature of asthma and likely contributes to the pathogenesis of airway hyperresponsiveness. We examined expression of genes related to airway wall fibrosis in a model of chronic allergen-induced airway dysfunction using laser capture microdissection and quantitative real-time PCR. BALB/c mice were sensitized and subjected to chronic ovalbumin exposure over a 12-wk period, after which they were rested and then harvested 2 and 8 wk after the last exposure. Chronic allergen-exposed mice had significantly increased indices of airway remodeling and airway hyperreactivity at all time points, although no difference in expression of fibrosis-related genes was found when mRNA extracted from whole lung was examined. In contrast, fibrosis-related gene expression was significantly upregulated in mRNA obtained from microdissected bronchial wall at 2 wk after chronic allergen exposure. In addition, when bronchial wall epithelium and smooth muscle were separately microdissected, gene expression of transforming growth factor-beta1 and plasminogen activating inhibitor-1 were significantly upregulated only in the airway epithelium. These data suggest that transforming growth factor-beta1 and other profibrotic mediators produced by airway wall, and specifically, airway epithelium, play an important role in the pathophysiology of airway remodeling.

  7. 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.

  8. Growth inhibition of Candida by human oral epithelial cells.

    PubMed

    Steele, C; Leigh, J; Swoboda, R; Fidel, P L

    2000-11-01

    Oropharyngeal candidiasis (OPC) caused by Candida albicans is a significant problem in human immunodeficiency virus (HIV)-infected persons. Recognizing the paucity of information on innate and/or adaptive mucosal host defenses against C. albicans, we recently reported that human and nonhuman primate and mouse vaginal epithelial cells inhibit the growth of C. albicans in vitro. In the present study, oral epithelial cells collected from saliva of healthy volunteers and a purified oral epithelial cell line were found to inhibit blastoconidia and/or hyphal growth of several Candida species. Cell contact was a strict requirement for the epithelial cell anti-Candida activity; neither saliva nor culture supernatants alone inhibited Candida growth, and addition of saliva to the coculture did not modulate the epithelial cell activity. Finally, epithelial cell anti-Candida activity was significantly lower in HIV-infected persons with OPC. Together, these results suggest that oral epithelial cells may play a role in innate resistance against OPC.

  9. Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit.

    PubMed

    von Moltke, Jakob; Ji, Ming; Liang, Hong-Erh; Locksley, Richard M

    2016-01-14

    Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells and smooth muscle hypercontractility. This response, known as 'weep and sweep', requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells). Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells. PMID:26675736

  10. Ionizing radiation induces heritable disruption of epithelial cell interactions

    PubMed Central

    Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2003-01-01

    Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, β-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell–cell communication, aberrant cell–extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization. PMID:12960393

  11. Perivascular mast cells regulate vein graft neointimal formation and remodeling

    PubMed Central

    Grassia, Gianluca; Cambrook, Helen; Ialenti, Armando; MacRitchie, Neil; Carberry, Jaclyn; Lawrence, Catherine

    2015-01-01

    Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method. Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient KitW−sh/W−sh mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in KitW−sh/W−sh mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice. Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling. PMID:26312183

  12. Chronic inflammatory cells with epithelial cell characteristics in teleost fishes.

    PubMed

    Noga, E J; Dykstra, M J; Wright, J F

    1989-09-01

    Certain cells that participate in the chronic inflammatory response of teleost fishes have many features typical of epithelioid cells of mammals. Such features include high metabolic activity, frequent phagolysosomes, and cytoplasmic interdigitations between adjacent cells; however, the epithelioid granulomas formed in response to certain diseases in teleost fishes also have several features associated with epithelial cells. Cases of ulcerative mycosis or acid-fast bacterial infection in Atlantic menhaden (Brevoortia tyrannus), fungal infection in silver perch (Bairdiella chrysoura), and mycobacteriosis in Mozambique tilapia (Oreochromis mossambicus) had epithelioid cells that were joined together by well-formed desmosomes with tonofilaments. "Mature granulomas" of the ulcerative mycosis-infected menhaden stained positively for cytokeratin, a cytoskeletal protein that is considered to be highly specific for epithelial cells. The consistent presence of these heretofore unrecognized epithelial features suggest that they may be characteristic of certain types of cells participating in piscine chronic inflammation. PMID:2686148

  13. 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

  14. Signalling from dead cells drives inflammation and vessel remodelling.

    PubMed

    Bennett, Martin; Yu, Haixiang; Clarke, Murray

    2012-01-01

    Death of vascular smooth muscle cells (VSMCs) has been demonstrated in vessel development and in disease, most notably in atherosclerosis, but also after injury and remodelling. VSMC death promotes multiple features of vulnerable plaques, but also induces features of normal vessel ageing and cystic medial necrosis, including loss of VSMCs, elastin fragmentation and loss, increased glycosaminoglycans and speckled calcification. VSMC apoptosis in the absence of efficient phagocytosis also produces inflammation due to secondary necrosis; in contrast, VSMC apoptosis in normal vessels can be silent. We have investigated the consequences of VSMC apoptosis in both disease and during vessel remodelling. We find that VSMCs release specific cytokines dependent upon the mode of cell death; IL-1β predominates during apoptosis, whilst IL-1α predominates during necrosis. Both IL-1α and β promote release of further cytokines from adjacent live cells, in particular IL-6 and MCP-1. The balance of cytokines results in pathology with differing compositions, including inflammation or neointima formation/vascular repair, via direct promotion of VSMC proliferation and migration. Thus, VSMC death can promote either pathology or repair, depending upon the context and cytokine signalling.

  15. 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

  16. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    PubMed Central

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

  17. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells.

    PubMed

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-11-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

  18. Cochlear outer hair cells undergo an apical circumference remodeling constrained by the hair bundle shape.

    PubMed

    Etournay, Raphaël; Lepelletier, Léa; Boutet de Monvel, Jacques; Michel, Vincent; Cayet, Nadège; Leibovici, Michel; Weil, Dominique; Foucher, Isabelle; Hardelin, Jean-Pierre; Petit, Christine

    2010-04-01

    Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.

  19. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    PubMed Central

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  20. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    PubMed Central

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

  1. Isolated Epithelial Cells of the Toad Bladder

    PubMed Central

    Gatzy, J. T.; Berndt, W. O.

    1968-01-01

    Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. QOO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O2 at a rate of 4 µl hr-1 dry wt-1. QOO2 was increased 50% by ADH (100 U/liter) or by cyclic 3',5'-AMP (10 mM/liter). Na+-free Ringer's depressed the QOO2 by 40%. The QOO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na+-free Ringer's but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na+ lack or ADH. The intracellular Na+ and K+ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H2O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na+ concentrations. Cells unresponsive to ADH and Na+ lack had high sucrose spaces and low transcellular membrane gradients of Na+, K+, and Cl-. The results suggest that trypsin and EDTA disaggregation damage the active Na+ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally. PMID:4300150

  2. Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling

    PubMed Central

    McClatchey, Shelly TH; Wang, Zheng; Linden, Lara M; Hastie, Eric L; Wang, Lin; Shen, Wanqing; Chen, Alan; Chi, Qiuyi; Sherwood, David R

    2016-01-01

    Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, we have discovered that the invasive uterine anchor cell activates Notch signaling in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, we found that Notch activation upregulates expression of ctg-1, which encodes a Sec14-GOLD protein, a member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, our results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues. DOI: http://dx.doi.org/10.7554/eLife.17218.001 PMID:27661254

  3. The Dynamics in Epithelial Cell Intercalation in Drosophila Morphogenesis

    NASA Astrophysics Data System (ADS)

    Wolf, Fred; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Eule, Stephan; Metzger, Jakob; Großhans, Jörg

    2015-03-01

    Epithelial cell rearrangement is important for many processes in morphogenesis. During germband extension in early gastrulation of Drosophila embryos, exchange of neighbors is achieved by junction remodeling that follows a topological T1 process. Its first step is the constriction of dorsal-ventral junctions and fusion of two 3x vertices into a 4x vertex a process believed to be junction autonomous. We established a high throughput imaging pipeline, by which we recorded, segmented and analysed more than 1000 neighbor exchanges in drosophila embryos. Characterizing the dynamics of junction lengths we find that the constriction of cell contacts follows intriguingly simple quantitative laws. (1) The mean contact length decreases approximately as a square root of time to collapse. (2) The time dependent variance of contact lengths is proportional to the square of the mean. (3) The time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations are sufficient to derive a stochastic differential equation for contact length that captures the non-equilibrium statistical mechanics of contact collapse. Supported by the German Research Foundation.

  4. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells

    PubMed Central

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-01-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell–cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell–cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett’s, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin–catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

  5. Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse

    PubMed Central

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

    2013-01-01

    The integrity and function of epithelial tissues depends 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. PMID:24095903

  6. Cell Death and Tissue Remodeling in Planarian Regeneration

    PubMed Central

    Pellettieri, Jason; Fitzgerald, Patrick; Watanabe, Shigeki; Mancuso, Joel; Green, Douglas R.; Alvarado, Alejandro Sánchez

    2010-01-01

    Many long-lived organisms, including humans, can regenerate some adult tissues lost to physical injury or disease. Much of the previous research on mechanisms of regeneration has focused on adult stem cells, which give rise to new tissue necessary for the replacement of missing body parts. Here we report that apoptosis of differentiated cells complements stem cell division during regeneration in the planarian Schmidtea mediterranea. Specifically, we developed a whole-mount TUNEL assay that allowed us to document two dramatic increases in the rate of apoptosis following amputation – an intial localized response near the wound site and a subsequent systemic response that varies in magnitude depending on the type of fragment examined. The latter cell death response can be induced in uninjured organs, occurs in the absence of planarian stem cells, and can also be triggered by prolonged starvation. Taken together, our results implicate apoptosis in the restoration of proper anatomical scale and proportion through remodeling of existing tissues. We also report results from initial mechanistic studies of apoptosis in planarians, which revealed that a S. mediterranea homolog of the antiapoptotic gene BCL2 is required for cell survival in adult animals. We propose that apoptosis is a central mechanism working in concert with stem cell division to restore anatomical form and function during metazoan regeneration. PMID:19766622

  7. 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.

  8. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G.; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-01-01

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  9. [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). PMID:27647251

  10. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

    PubMed

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Bou Dargham, Daria; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B Franklin; Gérard, Matthieu

    2016-02-01

    ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

  11. Avian prostatic acid phosphatase: estrogen regulation in the oviduct and epithelial cell-derived ovarian carcinomas.

    PubMed

    Bae, Hyocheol; Lim, Whasun; Bae, Seung-Min; Bazer, Fuller W; Choi, Youngsok; Song, Gwonhwa

    2014-07-01

    Prostatic acid phosphatase (ACPP) is a glycoprotein that is mainly synthesized and secreted by glandular epithelial cells (GE) of the prostate, and it is well known as a biomarker for prostate cancer. Although ACPP was used as prognostic/diagnostic indicator and studied to elucidate regulatory mechanism(s) during several decades in humans, its role is not clearly understood. Gene profiling data using a chicken DNA microarray revealed that ACPP increased significantly during remodeling and recrudescence of the oviduct in response to estrogen. Thus, in this study, we investigated the expression and hormonal regulation of ACPP gene in the reproductive tracts of chickens. ACPP was specifically detected in the luminal cells (LE) and GE of chicken oviduct, and diethylstilbestrol (a synthetic nonsteroidal estrogen) stimulated its expression during development of the oviduct. In addition, ACPP mRNA and protein were localized to LE and GE during the regeneration phase of the oviduct of laying hens during induced molting. Furthermore, ACPP mRNA and protein were abundant in GE of ovarian carcinoma, but not in normal ovaries. Moreover, strong expression of ACPP protein was detected in epithelial cells of cancerous ovaries from women. Collectively, results of the present study are the first to show that ACPP is a novel estrogen-stimulated gene in the oviductal epithelial cells of the chicken and that its expression increases significantly in epithelial cells of ovarian carcinoma, which indicates that it may be a candidate biomarker for diagnosis of epithelia-derived ovarian cancer in women. PMID:24829029

  12. 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

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

    PubMed

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

    2016-03-01

    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

  14. 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

  15. Characterization of glomerular epithelial cell matrix receptors.

    PubMed Central

    Adler, S.

    1992-01-01

    Integrin matrix receptors on glomerular epithelial cells (GEC) may play an important role in adhesion of GEC to the glomerular basement membrane (GBM) and in the maintenance of normal glomerular permeability. Therefore, the author determined the types of matrix receptors present on cultured rat GEC and examined their interactions with several components of the extracellular matrix. Beta 1 integrin matrix receptors were detected on all three glomerular cell types in rat kidney in vivo and at areas of cell-cell contact on cultured GEC. Glomerular epithelial cell adhesion to types I and IV collagen was slightly greater than to laminin and fibronectin. Adhesion to fibronectin was significantly inhibited by a synthetic peptide containing the RGD adhesion sequence. Immunoprecipitation of lysates of surface-iodinated GEC showed the presence of alpha 3 beta 1 integrin. Chromatography of lysates on immobilized collagen showed alpha 3 beta 1 integrin and a 70- to 75-kd protein band as the collagen receptors on GEC. Chromatography on the 120-kd cell-binding fragment of fibronectin disclosed only alpha 3 beta 1 as a specific fibronectin receptor. Antibody to the beta 1 integrin chain inhibited adhesion to laminin and collagen. These studies demonstrate that in vitro, as in vivo, GEC appear to express only alpha 3 beta 1 integrin. Furthermore, this matrix receptor is capable of mediating GEC adhesion to collagen, fibronectin, and laminin, components of the GBM, and presumably plays a similar role in promoting GEC adhesion to GBM in vivo. Images Figure 1 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1325740

  16. 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.

  17. 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.

  18. Anthrax Toxin Entry into Polarized Epithelial Cells

    PubMed Central

    Beauregard, Kathryn E.; Wimer-Mackin, Susan; Collier, R. John; Lencer, Wayne I.

    1999-01-01

    We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl− secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl− secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease. PMID:10338515

  19. Polarizing intestinal epithelial cells electrically through Ror2

    PubMed Central

    Cao, Lin; McCaig, Colin D.; Scott, Roderick H.; Zhao, Siwei; Milne, Gillian; Clevers, Hans; Zhao, Min; Pu, Jin

    2014-01-01

    ABSTRACT The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodeled the actin cytoskeleton selectively on the cathode side. In addition, an applied electric field also activated ERK1/2 and LKB1 (also known as STK11), key molecules in apical membrane formation. Disruption of the tyrosine protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly, and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous electric field created by the transepithelial potential difference might act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2–ERK signaling. PMID:24928904

  20. Polarizing intestinal epithelial cells electrically through Ror2.

    PubMed

    Cao, Lin; McCaig, Colin D; Scott, Roderick H; Zhao, Siwei; Milne, Gillian; Clevers, Hans; Zhao, Min; Pu, Jin

    2014-08-01

    The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodeled the actin cytoskeleton selectively on the cathode side. In addition, an applied electric field also activated ERK1/2 and LKB1 (also known as STK11), key molecules in apical membrane formation. Disruption of the tyrosine protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly, and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous electric field created by the transepithelial potential difference might act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2-ERK signaling. PMID:24928904

  1. 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

  2. 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

  3. Apoptosis during embryonic tissue remodeling is accompanied by cell senescence

    PubMed Central

    Lorda-Diez, Carlos I.; Garcia-Riart, Beatriz; Montero, Juan A.; Rodriguez-León, Joaquín; Garcia-Porrero, Juan A; Hurle, Juan M.

    2015-01-01

    This study re-examined the dying process in the interdigital tissue during the formation of free digits in the developing limbs. We demonstrated that the interdigital dying process was associated with cell senescence, as deduced by induction of β-gal activity, mitotic arrest, and transcriptional up-regulation of p21 together with many components of the senescence-associated secretory phenotype. We also found overlapping domains of expression of members of the Btg/Tob gene family of antiproliferative factors in the regressing interdigits. Notably, Btg2 was up-regulated during interdigit remodeling in species with free digits but not in the webbed foot of the duck. We also demonstrate that oxidative stress promoted the expression of Btg2, and that FGF2 and IGF1 which are survival signals for embryonic limb mesenchyme inhibited Btg2 expression. Btg2 overexpression in vivo and in vitro induced all the observed changes during interdigit regression, including oxidative stress, arrest of cell cycle progression, transcriptional regulation of senescence markers, and caspase-mediated apoptosis. Consistent with the central role of p21 on cell senescence, the transcriptional effects induced by overexpression of Btg2 are attenuated by silencing p21. Our findings indicate that cell senescence and apoptosis are complementary processes in the regression of embryonic tissues and share common regulatory signals. PMID:26568417

  4. 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

  5. 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

  6. 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

  7. Eikenella corrodens adherence to human buccal epithelial cells.

    PubMed Central

    Yamazaki, Y; Ebisu, S; Okada, H

    1981-01-01

    The mechanism of Eikenella corrodens adherence to human buccal epithelial cells in vitro was studied. Initial experiments to determine the optimal conditions for adherence of E. corrodens to buccal epithelial cells showed that adherence was dependent on time, temperature, bacterial concentration, and pH. Different strains of E. corrodens varied in their ability to adhere, and strain 1073 showed the greatest ability in adherence. Strain 1073 was selected for studies of adherence mechanisms. Trypsin treatment or heating (100 degrees C, 10 min) of the bacterial cells abolished their capacity to adhere to buccal epithelial cells. Treatment of buccal epithelial cells with trypsin also abolished adherence of E. corrodens 1073, whereas neuraminidase treatment of buccal epithelial cells enhanced the adherence. The adherence was inhibited by ethylenediaminetetraacetic acid and restored by adding Ca2+. The adherence was remarkably inhibited by sugars containing D-galactose and n-acetyl-D-galactosamine. Treatment of neuraminidase-treated epithelial cells with sodium metaperiodate or alpha- and beta-galactosidase did not decrease the adherence. These data suggest that adherence of E. corrodens 1073 to human buccal epithelial cells may require the interaction of lectin-like proteins on the bacterial surface with galactose-like receptors on the surface of epithelial cells. PMID:6260661

  8. Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

    PubMed

    Gerbe, François; Sidot, Emmanuelle; Smyth, Danielle J; Ohmoto, Makoto; Matsumoto, Ichiro; Dardalhon, Valérie; Cesses, Pierre; Garnier, Laure; Pouzolles, Marie; Brulin, Bénédicte; Bruschi, Marco; Harcus, Yvonne; Zimmermann, Valérie S; Taylor, Naomi; Maizels, Rick M; Jay, Philippe

    2016-01-14

    Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection. PMID:26762460

  9. 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

  10. Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys

    PubMed Central

    Ballinger, Carol A.; Plopper, Charles G.; McDonald, Ruth J.; Bartolucci, Alfred A.; Postlethwait, Edward M.; Harkema, Jack R.

    2011-01-01

    Children chronically exposed to high levels of ozone (O3), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O3 [0.5 parts per million (ppm), 8 h/day; “1-cycle”] or filtered air (FA) or 11 biweekly cycles of O3 (FA days 1–9; 0.5 ppm, 8 h/day on days 10–14; “11-cycle”). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH2), and uric acid (UA) concentration. Eleven-cycle O3 induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O3 also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments. PMID:21131400

  11. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form

    PubMed Central

    Sandoz, Kelsi M.; Popham, David L.; Beare, Paul A.; Sturdevant, Daniel E.; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A.

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3–3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3–3 cross-links as opposed to 16% 3–3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella’s environmental resistance. PMID:26909555

  12. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form.

    PubMed

    Sandoz, Kelsi M; Popham, David L; Beare, Paul A; Sturdevant, Daniel E; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance. PMID:26909555

  13. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

    PubMed Central

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Dargham, Daria Bou; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P.; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B. Franklin; Gérard, Matthieu

    2015-01-01

    Summary ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers1–3 target specific nucleosomes to regulate transcription is unclear. Here, we present genome-wide remodeller-nucleosome interaction profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank MNase-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites (TSSs) are nevertheless chromatinized with non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and modifications (H3K4me3 and H3K27ac). RNA polymerase (pol) II therefore navigates hundreds of bp of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. PMID:26814966

  14. Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells

    PubMed Central

    Robinson, Benjamin K.; Cortes, Ernesto; Rice, Alistair J.; Sarper, Muge

    2016-01-01

    ABSTRACT Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment. PMID:27170254

  15. 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

  16. Adipose Mesenchymal Stem Cell Secretome Modulated in Hypoxia for Remodeling of Radiation-Induced Salivary Gland Damage

    PubMed Central

    An, Hye-Young; Shin, Hyun-Soo; Choi, Jeong-Seok; Kim, Hun Jung

    2015-01-01

    Background and Purpose This study was conducted to determine whether a secretome from mesenchymal stem cells (MSC) modulated by hypoxic conditions to contain therapeutic factors contributes to salivary gland (SG) tissue remodeling and has the potential to improve irradiation (IR)-induced salivary hypofunction in a mouse model. Materials and Methods Human adipose mesenchymal stem cells (hAdMSC) were isolated, expanded, and exposed to hypoxic conditions (O2 < 5%). The hypoxia-conditioned medium was then filtered to a high molecular weight fraction and prepared as a hAdMSC secretome. The hAdMSC secretome was subsequently infused into the tail vein of C3H mice immediately after local IR once a day for seven consecutive days. The control group received equal volume (500 μL) of vehicle (PBS) only. SG function and structural tissue remodeling by the hAdMSC secretome were investigated. Human parotid epithelial cells (HPEC) were obtained, expanded in vitro, and then irradiated and treated with either the hypoxia-conditioned medium or a normoxic control medium. Cell proliferation and IR-induced cell death were examined to determine the mechanism by which the hAdMSC secretome exerted its effects. Results The conditioned hAdMSC secretome contained high levels of GM-CSF, VEGF, IL-6, and IGF-1. Repeated systemic infusion with the hAdMSC secretome resulted in improved salivation capacity and increased levels of salivary proteins, including amylase and EGF, relative to the PBS group. The microscopic structural integrity of SG was maintained and salivary epithelial (AQP-5), endothelial (CD31), myoepithelial (α-SMA) and SG progenitor cells (c-Kit) were successfully protected from radiation damage and remodeled. The hAdMSC secretome strongly induced proliferation of HPEC and led to a significant decrease in cell death in vivo and in vitro. Moreover, the anti-apoptotic effects of the hAdMSC secretome were found to be promoted after hypoxia-preconditioning relative to normoxia

  17. 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

  18. 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

  19. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  20. 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. PMID:26641038

  1. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

    PubMed

    Le Guen, Ludovic; Marchal, Stéphane; Faure, Sandrine; de Santa Barbara, Pascal

    2015-10-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  2. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    PubMed

    Knodler, Leigh A; Nair, Vinod; Steele-Mortimer, Olivia

    2014-01-01

    Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  3. Transforming Growth Factor β/activin signalling induces epithelial cell flattening during Drosophila oogenesis

    PubMed Central

    Brigaud, Isabelle; Duteyrat, Jean-Luc; Chlasta, Julien; Le Bail, Sandrine; Couderc, Jean-Louis; Grammont, Muriel

    2015-01-01

    ABSTRACT Although the regulation of epithelial morphogenesis is essential for the formation of tissues and organs in multicellular organisms, little is known about how signalling pathways control cell shape changes in space and time. In the Drosophila ovarian epithelium, the transition from a cuboidal to a squamous shape is accompanied by a wave of cell flattening and by the ordered remodelling of E-cadherin-based adherens junctions. We show that activation of the TGFβ pathway is crucial to determine the timing, the degree and the dynamic of cell flattening. Within these cells, TGFβ signalling controls cell-autonomously the formation of Actin filament and the localisation of activated Myosin II, indicating that internal forces are generated and used to remodel AJ and to promote cytoskeleton rearrangement. Our results also reveal that TGFβ signalling controls Notch activity and that its functions are partly executed through Notch. Thus, we demonstrate that the cells that undergo the cuboidal-to-squamous transition produce active cell-shaping mechanisms, rather than passively flattening in response to a global force generated by the growth of the underlying cells. Thus, our work on TGFβ signalling provides new insights into the mechanisms through which signal transduction cascades orchestrate cell shape changes to generate proper organ structure. PMID:25681395

  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. PMID:25506353

  6. 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.

  7. Bone marrow-derived cells participate in stromal remodeling of the lung following acute bacterial pneumonia in mice.

    PubMed

    Serikov, Vladimir B; Mikhaylov, Viatcheslav M; Krasnodembskay, Anna D; Matthay, Michael A

    2008-01-01

    Bone marrow-derived cells (BMDC) have been shown to graft injured tissues, differentiate in specialized cells, and participate in repair. The importance of these processes in acute lung bacterial inflammation and development of fibrosis is unknown. The goal of this study was to investigate the temporal sequence and lineage commitment of BMDC in mouse lungs injured by bacterial pneumonia. We transplanted GFP-tagged BMDC into 5-Gy-irradiated C57BL/6 mice. After 3 months of recovery, mice were subjected to LD(50) intratracheal instillation of live E. coli (controls received saline) which produced pneumonia and subsequent areas of fibrosis. Lungs were investigated by immunohistology for up to 6 months. At the peak of lung inflammation, the predominant influx of BMDC were GFP(+) leukocytes. Postinflammatory foci of lung fibrosis were evident after 1-2 months. The fibrotic foci in lung stroma contained clusters of GFP(+) CD45(+) cells, GFP(+) vimentin-positive cells, and GFP(+) collagen I-positive fibroblasts. GFP(+) endothelial or epithelial cells were not identified. These data suggest that following 5-Gy irradiation and acute bacterial pneumonia, BMDC may temporarily participate in lung postinflammatory repair and stromal remodeling without long-term engraftment as specialized endothelial or epithelial cells.

  8. Regenerative potential of human schneiderian membrane: progenitor cells and epithelial-mesenchymal transition.

    PubMed

    Derjac-Aramă, A I; Sarafoleanu, C; Manea, C M; Nicolescu, M I; Vrapciu, A D; Rusu, M C

    2015-12-01

    An innate osteogenic potential of the Schneiderian membrane (SM) is progressively assessed in studies ranging from non-human species to human subjects. It has relevance for endosteal placement and osseointegration. Nestin-expressing osteogenic progenitor cells are allegedly involved in bone formation and remodelling. Nestin phenotype was not assessed previously in human SM. We therefore aimed to fill that particular gap in the literature. Bioptic samples of human adult SM were obtained during surgery from eight adult patients, operated for non-malignant pathologies. Immunohistochemistry on paraffin-embedded tissue samples used primary antibodies against nestin, CD45, CD146, cytokeratin 7 (CK7), and alpha-smooth muscle actin (α-SMA). Nestin expression was consistently found in endothelial cells, and was scarcely encountered in pericytes, putative stromal stem/progenitor cells, as well as in glandular epithelial cells. Moreover, woven bone formation in the periosteal layer of the SM can also be regarded as evidence of the osteogenic potential of this membrane. Nestin and CD45 expression in cells of the primary bone supports the osteogenic potential of SM nestin-expressing cells and a possible involvement of hematopoietic stem cells in maxillary sinus floor remodeling. CD146, a known inducer of epithelial-mesenchymal transition (EMT), was expressed in epithelia, as was CK7. Isolated stromal cells were found expressing CD146, CK7 and α-SMA, suggesting that regenerative processes happening in the SM may also involve processes of EMT which generate stem/progenitor cells. This study provides additional evidence for the regenerative potential of the Schneiderian membrane and identifies potential roles for cells of its stem niche in osteogenesis. PMID:26414809

  9. 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

  10. Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells.

    PubMed

    Koo, Michael Jakun; Rooney, Kristen T; Choi, Mary E; Ryter, Stefan W; Choi, Augustine M K; Moon, Jong-Seok

    2015-08-28

    Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells.

  11. 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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23814196

  13. Myocardial remodeling in diabetic cardiomyopathy associated with cardiac mast cell activation.

    PubMed

    Huang, Zhi Gang; Jin, Qun; Fan, Min; Cong, Xiao Liang; Han, Shu Fang; Gao, Hai; Shan, Yi

    2013-01-01

    Diabetic cardiomyopathy is a specific disease process distinct from coronary artery disease and hypertension. The disease features cardiac remodeling stimulated by hyperglycemia of the left ventricle wall and disrupts contractile functions. Cardiac mast cells may be activated by metabolic byproducts resulted from hyperglycermia and then participate in the remodeling process by releasing a multitude of cytokines and bioactive enzymes. Nedocromil, a pharmacologic stabilizer of mast cells, has been shown to normalize cytokine levels and attenuate cardiac remodeling. In this study, we describe the activation of cardiac mast cells by inducing diabetes in normal mice using streptozotocin (STZ). Next, we treated the diabetic mice with nedocromil for 12 weeks and then examined their hearts for signs of cardiac remodeling and quantified contractile function. We observed significantly impaired heart function in diabetic mice, as well as increased cardiac mast cell density and elevated mast cell secretions that correlated with gene expression and aberrant cytokine levels associated with cardiac remodeling. Nedocromil treatment halted contractile dysfunction in diabetic mice and reduced cardiac mast cell density, which correlated with reduced bioactive enzyme secretions, reduced expression of extracellular matrix remodeling factors and collagen synthesis, and normalized cytokine levels. However, the results showed nedocromil treatments did not return diabetic mice to a normal state. We concluded that manipulation of cardiac mast cell function is sufficient to attenuate cardiomyopathy stimulated by diabetes, but other cellular pathways also contribute to the disease process.

  14. 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

  15. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

  16. Protein kinase D is increased and activated in lung epithelial cells and macrophages in idiopathic pulmonary fibrosis.

    PubMed

    Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.

  17. Crystal violet staining to quantify Candida adhesion to epithelial cells.

    PubMed

    Negri, M; Gonçalves, V; Silva, S; Henriques, M; Azeredo, J; Oliveira, R

    2010-01-01

    In vitro studies of adhesion capability are essential to characterise the virulence of Candida species. However, the assessment of adhesion by traditional methods is time-consuming. The aim of the present study is the development of a simple methodology using crystal violet staining to quantify in vitro adhesion of different Candida species to epithelial cells. The experiments are performed using Candida albicans (ATCC 90028), C. glabrata (ATCC 2001), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750). A human urinary bladder epithelial cell line (TCC-SUP) is used. Yeast and epithelial cells were stained with crystal violet, epithelial cells were then destained using intermediate washing, and the dye in the yeast cells was extracted with acetic acid. The method was validated for the different Candida reference species by comparison with traditional microscope observation and enumeration. The method was then used to assess Candida adhesion to epithelial cells and also to silicone. For all Candida spp. high correlation values (r2= 0.9724-0.9997) between the number of adherent yeasts (microscope enumeration) and absorbance values were obtained for an inoculum concentration >10(6) cells/mL. The proposed technique was easy to perform and reproducible, enabling the determination of adhesion ability of Candida species to an epithelial cell line. PMID:20973406

  18. 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.

  19. 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.

  20. 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. PMID:20400167

  1. 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.

  2. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    SciTech Connect

    Ostrup, Olga; Hyttel, Poul; Klaerke, Dan A.; Collas, Philippe

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  3. Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis.

    PubMed

    Khosravi, Farhad; Michel, Vera; Galuska, Christina E; Bhushan, Sudhanshu; Christian, Philipp; Schuppe, Hans-Christian; Pilatz, Adrian; Galuska, Sebastian P; Meinhardt, Andreas

    2016-08-19

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis. PMID:27339898

  4. Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells

    PubMed Central

    Andersen, Nicholas J.

    2010-01-01

    The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains. PMID:19889837

  5. ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion

    PubMed Central

    Chronopoulos, Antonios; Robinson, Benjamin; Sarper, Muge; Cortes, Ernesto; Auernheimer, Vera; Lachowski, Dariusz; Attwood, Simon; García, Rebeca; Ghassemi, Saba; Fabry, Ben; del Río Hernández, Armando

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-dependent downregulation of actomyosin (MLC-2) contractility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. Our findings implicate a RAR-β/MLC-2 pathway in peritumoural stromal remodelling and mechanosensory-driven activation of PSCs, and further suggest that mechanical reprogramming of PSCs with retinoic acid derivatives might be a viable alternative to stromal ablation strategies for the treatment of PDAC. PMID:27600527

  6. ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion.

    PubMed

    Chronopoulos, Antonios; Robinson, Benjamin; Sarper, Muge; Cortes, Ernesto; Auernheimer, Vera; Lachowski, Dariusz; Attwood, Simon; García, Rebeca; Ghassemi, Saba; Fabry, Ben; Del Río Hernández, Armando

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-dependent downregulation of actomyosin (MLC-2) contractility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. Our findings implicate a RAR-β/MLC-2 pathway in peritumoural stromal remodelling and mechanosensory-driven activation of PSCs, and further suggest that mechanical reprogramming of PSCs with retinoic acid derivatives might be a viable alternative to stromal ablation strategies for the treatment of PDAC. PMID:27600527

  7. Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model

    PubMed Central

    Juuti-Uusitalo, Kati M; Kaukinen, Katri; Mäki, Markku; Tuimala, Jarno; Kainulainen, Heikki

    2006-01-01

    Background The TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. Results The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. Conclusion Our results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells. PMID:17074098

  8. Influence of the mesenchymal cell source on oral epithelial development.

    PubMed

    Kinikoglu, Beste; Rovere, Marie Rose; Haftek, Marek; Hasirci, Vasif; Damour, Odile

    2012-03-01

    The extent of the influence of mesenchymal tissue on epithelial development is still debated, and elucidation of epithelial-mesenchymal interactions should be of relevance for controlling normal as well as pathological growth and development. The aim of the present study was to elucidate the influence of the mesenchymal cell type on oral mucosa epithelial development in vitro, using tissue-engineering principles, by including three different sources for mesenchymal cell type, viz. oral mucosa, skin and cornea, each of them presenting a distinct type of epithelium in situ. We investigated epithelial-mesenchymal interactions, considering both morphological criteria and protein expression (filaggrin, keratin 10, keratin 12, keratin 13 and laminin 5). The results of the histology, immunohistochemistry and transmission electron microscopy of the three types of tissue-engineered constructs composed of mesenchymal cells of different sources (oral, dermal and corneal fibroblasts) and of the same oral epithelial cells showed that the mesenchymal cell source had a significant influence on the thickness and ultrastructure of the epithelium, but not on the differentiation of oral epithelial cells, which might be an intrinsic property of these cells due to their genetic programming. PMID:21548135

  9. Isolation and characterization of cutaneous epithelial stem cells

    PubMed Central

    Jensen, Uffe B.; Ghazizadeh, Soosan; Owens, David M.

    2014-01-01

    SUMMARY During homeostasis, adult mammalian skin turnover is maintained by a number of multipotent and unipotent epithelial progenitors located either in the epidermis, hair follicle or sebaceous gland. Recent work has illustrated that these various progenitor populations reside in regionalized niches and are phenotypically distinct from one another. This degree of heterogeneity within the progenitor cell landscape in the cutaneous epithelium complicates our ability to target, purify and manipulate cutaneous epithelial stem cell subpopulations in adult skin. The techniques outlined in this chapter describe basic procedures for the isolation and purification of murine epithelial progenitors and assessing their capacity for ex vivo propagation. PMID:23483387

  10. TSLP in Epithelial Cell and Dendritic Cell Cross Talk

    PubMed Central

    Liu, Yong-Jun

    2013-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells that have the ability to sense infection and tissue stress, sample and present antigen to T lymphocytes, and instruct the initiation of different forms of immunity and tolerance. The functional versatility of DCs depends on their remarkable ability to translate collectively the information from the invading microbes, as well as their resident tissue microenvironments. Recent progress in understanding Toll-like receptor (TLR) biology has illuminated the mechanisms by which DCs link innate and adaptive antimicrobial immune responses. However, how tissue microenvironments shape the function of DCs has remained elusive. Recent studies of TSLP (thymic stromal lymphopoietin), an epithelial cell-derived cytokine that strongly activates DCs, provide strong evidence at a molecular level that epithelial cells/tissue microenvironments directly communicate with DCs, the professional antigen-presenting cells of the immune system. We review recent progress on how TSLP expressed within thymus and peripheral lymphoid and nonlymphoid tissues regulates DC-mediated central tolerance, peripheral T cell homeostasis, and inflammatory Th2 responses. PMID:19231591

  11. Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation during Acinar Differentiation

    PubMed Central

    Williams, Courtney M.; Engler, Adam J.; Slone, R. Daniel; Galante, Leontine L.; Schwarzbauer, Jean E.

    2009-01-01

    The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland. PMID:18451144

  12. Biliary epithelial cells proliferate during oxygenated ex situ liver culture

    PubMed Central

    Bian, Congwen; Du, Yiqi; Ding, Rui; Huang, Jun; Dai, Yan; Bao, Sujin; Zhao, Lijuan; Shen, Hefang; Dong, Jing; Xu, Jianjian; Xiong, Qiru; Xu, Lili

    2016-01-01

    Biliary complications remain a major source of morbidity in liver transplant patients. Among these complications, nonanastomotic biliary strictures (NAS) are especially common and they are frequently therapy resistant in part because biliary epithelial cells are more sensitive to warm ischemic injury than hepatocytes. It has been a challenge to maintain the physiological function of biliary epithelial cells during liver transplantation. In this work, we have examined the effect of oxygen on proliferation of biliary epithelial cells in the rat livers obtained from donation after circulatory death (DCD). Twelve rat livers from DCD were divided into two groups. Livers in the control group were isolated following a standard procedure without oxygen supply. Livers in the experimental group were isolated with a constant supply of oxygen. All livers were then connected to an ex situ liver culture system in the presence of bromodeoxyuridine (BrdU), a thymidine analogue and a marker for cell proliferation. After 6 hours of normothermic ex situ liver culture, morphology and DNA replication in hepatocytes and biliary epithelial cells were assessed and compared between the two groups. We found that about 4.5% of the biliary epithelial cells in the experimental group proliferated compared with only 0.4% of cells in the control based on BrdU staining. No significant change in cell morphology was observed in those cells between the two groups. Thus, our results indicate that oxygen supply is required for maintenance of the physiological function of biliary epithelial cells during liver transplant and suggest that a constant oxygen supply during liver isolation along with ex situ liver organ culture can enhance the repair of biliary epithelial cell injury during liver transplantation. PMID:27725875

  13. 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.

  14. 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.

  15. 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.

  16. Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

    SciTech Connect

    Gao, Fu; Chambon, Pierre; Tellides, George; Kong, Wei; Zhang, Xiaoming; Li, Wei

    2014-11-07

    Highlights: • TGF-β signaling in SMC contributes to the flow-induced vascular remodeling. • Disruption of TGF-β signaling in SMC can prevent this process. • Targeting SM-specific Tgfbr2 could be a novel therapeutic strategy for vascular remodeling. - Abstract: Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2{sup f/f}) and their corresponding wild-type background mice (MyhCre.Tgfbr2{sup WT/WT}) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.

  17. How Shigella Utilizes Ca(2+) Jagged Edge Signals during Invasion of Epithelial Cells.

    PubMed

    Bonnet, Mariette; Tran Van Nhieu, Guy

    2016-01-01

    Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca(2+) responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca(2+) increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca(2+) overload. In this review, we will focus on the role of Ca(2+) responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

  18. New aspects of vascular remodelling: the involvement of all vascular cell types.

    PubMed

    McGrath, John C; Deighan, Clare; Briones, Ana M; Shafaroudi, Majid Malekzadeh; McBride, Melissa; Adler, Jeremy; Arribas, Silvia M; Vila, Elisabet; Daly, Craig J

    2005-07-01

    Conventionally, the architecture of arteries is based around the close-packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self-maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re-arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.

  19. Role of autophagy in the regulation of epithelial cell junctions.

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  20. 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. PMID:20113446

  1. 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.

  2. 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.

  3. Trefoil peptides promote restitution of wounded corneal epithelial cells.

    PubMed

    Göke, M N; Cook, J R; Kunert, K S; Fini, M E; Gipson, I K; Podolsky, D K

    2001-04-01

    The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

  4. 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.

  5. Interplay of cell dynamics and epithelial tension during morphogenesis of the Drosophila pupal wing

    PubMed Central

    Etournay, Raphaël; Popović, Marko; Merkel, Matthias; Nandi, Amitabha; Blasse, Corinna; Aigouy, Benoît; Brandl, Holger; Myers, Gene; Salbreux, Guillaume; Jülicher, Frank; Eaton, Suzanne

    2015-01-01

    How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing. DOI: http://dx.doi.org/10.7554/eLife.07090.001 PMID:26102528

  6. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

    PubMed

    Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

    2015-11-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  7. Lingual Epithelial Stem Cells and Organoid Culture of Them.

    PubMed

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-28

    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.

  8. 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.

  9. Development of human epithelial cell systems for radiation risk assessment.

    PubMed

    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-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. PMID:11538024

  10. 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.

  11. Autophagic and apoptotic cell death in amniotic epithelial cells.

    PubMed

    Shen, Z-Y; Li, E-M; Lu, S-Q; Shen, J; Cai, Y-M; Wu, Y-E; Zheng, R-M; Tan, L-J; Xu, L-Y

    2008-11-01

    The aim of this paper is to determine if autophagic cell death is associated with apoptosis and whether it participates in the process of term amniotic rupture. Forty pieces of fresh term amnions, including twenty from a position near the margin of the placentas and twenty from the margin of the naturally ruptured part of the placentas in term gestation were collected, respectively. The amnions were examined by scanning electron microscopy (SEM) and amniotic epithelial (AE) cells were examined by transmission electron microscopy (TEM). Autophagic and apoptotic cell death (PCD) were assayed by laser scanning confocal microscopy (LSCM) or flow cytometry using monodansylcadaverin (MDC) and propidium iodide (PI) stain. BCL(2) and BAX were examined by immunoblotting. Under SEM the amniotic epithelia appeared normal in the position near the placenta. They had an atrophied appearance in the margin of their natural broken parts. In the AE cells PCD was divided into three subtypes by TEM: autophagic cell death with positive stains of MDC and PI; apoptotic cell death; and the mixed type. Quantitative detection showed that there were more death cells, including autophagic and apoptotic, in the AE cells near the ruptured parts than near the placentas. An increased expression of BAX and a decreased expression of BCL(2) protein in the AE cells near the broken margin were observed. Apoptotic and autophagic cell death by the intrinsic pathway are the basic event in the AE cell and they are involved in the cause of membrane rupture of the human amnion in term gestation.

  12. Transdifferentiation of endothelial cells to smooth muscle cells play an important role in vascular remodelling

    PubMed Central

    Coll-Bonfill, Núria; Musri, Melina Mara; Ivo, Victor; Barberà, Joan Albert; Tura-Ceide, Olga

    2015-01-01

    Pulmonary artery remodelling it is a major feature of pulmonary hypertension (PH). It is characterised by cellular and structural changes of the pulmonary arteries causing higher pulmonar vascular resistance and right ventricular failure. Abnormal deposition of smooth muscle-like (SM-like) cells in normally non-muscular, small diameter vessels and a deregulated control of endothelial cells are considered pathological features of PH. The origin of the SM-like cells and the mechanisms underlying the development and progression of this remodelling process are not understood. Endothelial cells within the intima may migrate from their organised layer of cells and transition to mesenchymal or SM-like phenotype in a process called endothelial-mesenchymal transition (EnMT). Traditionally, Waddington’s epigenetic landscape illustrates that fates of somatic cells are progressively determined to compulsorily follow a downhill differentiation pathway. EnMT induces the transformation of cells with stem cell traits, therefore contrasting Waddington’s theory and confirming that cell fate seems to be far more flexible than previously thought. The prospect of therapeutic inhibition of EnMT to delay or prevent PH may represent a promising new treatment modality. PMID:25973327

  13. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    PubMed

    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy.

  14. 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.

  15. Trans-sialidase Stimulates Eat Me Response from Epithelial Cells

    PubMed Central

    Butler, Claire E; de Carvalho, Tecia M U; Grisard, Edmundo C; Field, Robert A; Tyler, Kevin M

    2013-01-01

    Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake of microparticles into a perinuclear compartment in a manner which may emulate entosis. PMID:23601193

  16. 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

  17. 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

  18. Cell division and the maintenance of epithelial order

    PubMed Central

    Ragkousi, Katerina

    2014-01-01

    Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis. PMID:25349258

  19. Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.

    PubMed

    Nakahashi-Oda, Chigusa; Udayanga, Kankanam Gamage Sanath; Nakamura, Yoshiyuki; Nakazawa, Yuta; Totsuka, Naoya; Miki, Haruka; Iino, Shuichi; Tahara-Hanaoka, Satoko; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira

    2016-04-01

    Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-β (IFN-β), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-β production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis. PMID:26855029

  20. Bronchial epithelial cells: The key effector cells in the pathogenesis of chronic obstructive pulmonary disease?

    PubMed

    Gao, Wei; Li, Lingling; Wang, Yujie; Zhang, Sini; Adcock, Ian M; Barnes, Peter J; Huang, Mao; Yao, Xin

    2015-07-01

    The primary function of the bronchial epithelium is to act as a defensive barrier aiding the maintenance of normal airway function. Bronchial epithelial cells (BEC) form the interface between the external environment and the internal milieu, making it a major target of inhaled insults. However, BEC can also serve as effectors to initiate and orchestrate immune and inflammatory responses by releasing chemokines and cytokines, which recruit and activate inflammatory cells. They also produce excess reactive oxygen species as a result of an oxidant/antioxidant imbalance that contributes to chronic pulmonary inflammation and lung tissue damage. Accumulated mucus from hyperplastic BEC obstructs the lumen of small airways, whereas impaired cell repair, squamous metaplasia and increased extracellular matrix deposition underlying the epithelium is associated with airway remodelling particularly fibrosis and thickening of the airway wall. These alterations in small airway structure lead to airflow limitation, which is critical in the clinical diagnosis of chronic obstructive pulmonary disease (COPD). In this review, we discuss the abnormal function of BEC within a disturbed immune homeostatic environment consisting of ongoing inflammation, oxidative stress and small airway obstruction. We provide an overview of recent insights into the function of the bronchial epithelium in the pathogenesis of COPD and how this may provide novel therapeutic approaches for a number of chronic lung diseases.

  1. Angiomyolipoma with epithelial cysts: mimic of renal cell carcinoma.

    PubMed

    Rosenkrantz, Andrew B; Hecht, Elizabeth M; Taneja, Samir S; Melamed, Jonathan

    2010-01-01

    Angiomyolipoma with epithelial cysts (AMLEC) is a rare variant of angiomyolipoma with minimal fat that contains epithelial-lined cysts and may mimic a cystic renal cell carcinoma. While 17 cases have been described in the pathology literature since this entity was first described in 2006, the radiologic appearance was not demonstrated in any of these cases. We report the CT and MRI appearance of AMLEC found incidentally in a patient with lupus nephritis.

  2. RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells

    PubMed Central

    Akin, Debra; Newman, Jeremy R.B.; McIntyre, Lauren M.

    2016-01-01

    Purpose The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. Methods Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events. Results Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells and of the shared exon-skipping events, 92% of those differentially expressed were more frequent in the PNN knockdown. Conclusions These data suggest that lowering of PNN levels in

  3. Change in Cell Shape Is Required for Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition of Mammary Epithelial Cells

    PubMed Central

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2010-01-01

    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-β-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. PMID:18506791

  4. 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.

  5. Epithelial cell proliferation in nasal polyps could be up-regulated by platelet-derived growth factor.

    PubMed

    Coste, A; Wang, Q P; Roudot-Thoraval, F; Chapelin, C; Bedbeder, P; Poron, F; Peynègre, R; Escudier, E

    1996-05-01

    The modifications of epithelial differentiation and proliferation observed in nasal polyps (NP) could be related to local secretion of growth factors, among which platelet-derived growth factor (PDGF) could play a key role. We therefore prospectively studied, by immunohistochemistry, proliferating cell nuclear antigen (PCNA, an S-phase cell marker), PDGF, and CD-68 (activated macrophages marker) expression in NP and inferior turbinate mucosa (NM) in 11 patients. Our data show that PCNA and PDGF expression are increased in NP epithelium, while CD-68 expression is increased in NP epithelium and lamina propria when compared to NM. Increased local PDGF secretion by numerous activated macrophages could therefore be involved in epithelial cell proliferation up-regulation in NP. PDGF could also be involved in the pathogenesis of NP via its connective tissue remodeling actions.

  6. Primary human bronchial epithelial cells grown from explants.

    PubMed

    Yaghi, Asma; Zaman, Aisha; Dolovich, Myrna

    2010-01-01

    Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly

  7. Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells

    PubMed Central

    Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

    2016-01-01

    One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg–Gly–Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration. PMID:27733898

  8. Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study.

    PubMed

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins. PMID:23251682

  9. Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study.

    PubMed

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins.

  10. The Effects of Matrix Metalloproteinase-9 on Dairy Goat Mastitis and Cell Survival of Goat Mammary Epithelial Cells.

    PubMed

    Li, Hui; Zheng, Huiling; Li, Lihui; Shen, Xingai; Zang, Wenjuan; Sun, Yongsen

    2016-01-01

    Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent enzyme, and plays a crucial role in extracellular matrix degeneration, inflammation and tissue remodeling. However, the relationship between MMP-9 and somatic cell count (SCC) in goat milk and the role of MMP-9 in the regulation of mastitis are still unknown. In this study, we found MMP-9 was predominantly expressed in the spleen, intestine and mammary gland. The SCC in goat milk was positively correlated with MMP-9 expression, and staphylococcus aureus could markedly increase MMP-9 expression in goat mammary epithelial cells (GMEC) in dosage and time dependent manner. We also demonstrated that SB-3CT, an inhibitor of MMP-9, promoted apoptosis and inhibited proliferation in GMEC. Thus, MMP-9 may emerge as an easily measurable and sensitive parameter that reflects the number of somatic cells present in milk and a regulatory factor of apoptosis in GMEC. PMID:27518717

  11. The Effects of Matrix Metalloproteinase-9 on Dairy Goat Mastitis and Cell Survival of Goat Mammary Epithelial Cells

    PubMed Central

    Li, Hui; Zheng, Huiling; Li, Lihui; Shen, Xingai; Zang, Wenjuan; Sun, Yongsen

    2016-01-01

    Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent enzyme, and plays a crucial role in extracellular matrix degeneration, inflammation and tissue remodeling. However, the relationship between MMP-9 and somatic cell count (SCC) in goat milk and the role of MMP-9 in the regulation of mastitis are still unknown. In this study, we found MMP-9 was predominantly expressed in the spleen, intestine and mammary gland. The SCC in goat milk was positively correlated with MMP-9 expression, and staphylococcus aureus could markedly increase MMP-9 expression in goat mammary epithelial cells (GMEC) in dosage and time dependent manner. We also demonstrated that SB-3CT, an inhibitor of MMP-9, promoted apoptosis and inhibited proliferation in GMEC. Thus, MMP-9 may emerge as an easily measurable and sensitive parameter that reflects the number of somatic cells present in milk and a regulatory factor of apoptosis in GMEC. PMID:27518717

  12. Polarized distribution of IQGAP proteins in gastric parietal cells and their roles in regulated epithelial cell secretion.

    PubMed

    Zhou, Rihong; Guo, Zhen; Watson, Charles; Chen, Emily; Kong, Rong; Wang, Wenxian; Yao, Xuebiao

    2003-03-01

    Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.

  13. The TNF Family Molecules LIGHT and Lymphotoxin αβ Induce a Distinct Steroid-Resistant Inflammatory Phenotype in Human Lung Epithelial Cells.

    PubMed

    da Silva Antunes, Ricardo; Madge, Lisa; Soroosh, Pejman; Tocker, Joel; Croft, Michael

    2015-09-01

    Lung epithelial cells are considered important sources of inflammatory molecules and extracellular matrix proteins that contribute to diseases such as asthma. Understanding the factors that stimulate epithelial cells may lead to new insights into controlling lung inflammation. This study sought to investigate the responsiveness of human lung epithelial cells to the TNF family molecules LIGHT and lymphotoxin αβ (LTαβ). Bronchial and alveolar epithelial cell lines, and primary human bronchial epithelial cells, were stimulated with LIGHT and LTαβ, and expression of inflammatory cytokines and chemokines and markers of epithelial-mesenchymal transition and fibrosis/remodeling was measured. LTβ receptor, the receptor shared by LIGHT and LTαβ, was constitutively expressed on all epithelial cells. Correspondingly, LIGHT and LTαβ strongly induced a limited but highly distinct set of inflammatory genes in all epithelial cells tested, namely the adhesion molecules ICAM-1 and VCAM-1; the chemokines CCL5, CCL20, CXCL1, CXCL3, CXCL5, and CXCL11; the cytokines IL-6, activin A and GM-CSF; and metalloproteinases matrix metalloproteinase-9 and a disintegrin and metalloproteinase domain-8. Importantly, induction of the majority of these inflammatory molecules was insensitive to the suppressive effects of the corticosteroid budesonide. LIGHT and LTαβ also moderately downregulated E-cadherin, a protein associated with maintaining epithelial integrity, but did not significantly drive production of extracellular matrix proteins or α-smooth muscle actin. Thus, LIGHT and LTαβ induce a distinct steroid-resistant inflammatory signature in airway epithelial cells via constitutively expressed LTβ receptor. These findings support our prior murine studies that suggested the receptors for LIGHT and LTαβ contribute to development of lung inflammation characteristic of asthma and idiopathic pulmonary fibrosis.

  14. Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype

    PubMed Central

    Coll-Bonfill, Núria; Peinado, Victor I.; Pisano, María V.; Párrizas, Marcelina; Blanco, Isabel; Evers, Maurits; Engelmann, Julia C.; García-Lucio, Jessica; Tura-Ceide, Olga; Meister, Gunter

    2016-01-01

    Objective Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling. Methods and Results Slug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries. Conclusions Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases. PMID:27441378

  15. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  16. Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

    PubMed Central

    Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

    2012-01-01

    Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

  17. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells.

    PubMed Central

    Paris, S; Boisvieux-Ulrich, E; Crestani, B; Houcine, O; Taramelli, D; Lombardi, L; Latgé, J P

    1997-01-01

    The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. PMID:9119494

  18. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  19. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

    Plikus, Maksim V.; Gay, Denise L.; Treffeisen, Elsa; Wang, Anne; Supapannachart, Rarinthip June; Cotsarelis, George

    2012-01-01

    Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis hasa mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new inter-follicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover cellular and signaling basis of this remarkable adult wound regeneration phenomenon. PMID:23085626

  20. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

    PubMed Central

    St Johnston, Daniel

    2016-01-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside of the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  1. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  2. Characterization of discrete equine intestinal epithelial cell lineages

    PubMed Central

    Gonzalez, Liara M.; Kinnin, Leslie A.; Blikslager, Anthony T.

    2015-01-01

    OBJECTIVE To characterize epithelial cells of the small intestine and colon in horses without clinical gastrointestinal abnormalities with an emphasis on the stem cell niche constituents. SAMPLE Mucosal biopsy specimens from small and large intestines obtained from 12 horses euthanized for reasons unrelated to gastrointestinal disease or systemic disease. PROCEDURES Intestinal biopsy specimens were collected by sharp dissection immediately following euthanasia. Specimens were prepared for immunohistochemical, immunofluorescence, and transmission electron microscopic imaging to detect and characterize each epithelial cell type. Antibodies against protein biomarkers for cellular identification were selected on the basis of expression in other mammalian species. RESULTS Intestinal epithelial cell types were identified by means of immunostaining and morphological characterization with transmission electron microscopy. Some differences in biomarker expression and antibody cross-reactivity were identified in equine tissue, compared with other species. However, each known type of mucosal epithelial cell was identified in equine tissue. CONCLUSIONS AND CLINICAL RELEVANCE The methodology used can enhance detection of stem cells and progenitor cells as well as postmitotic cell lineages in equine intestinal tissues. Results may have relevance to regenerative potential of intestinal mucosa and survival in horses with colic. PMID:25815577

  3. Nipah Virus Entry and Egress from Polarized Epithelial Cells

    PubMed Central

    Lamp, Boris; Dietzel, Erik; Kolesnikova, Larissa; Sauerhering, Lucie; Erbar, Stephanie; Weingartl, Hana

    2013-01-01

    Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal. PMID:23283941

  4. Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

    PubMed

    Lange-Consiglio, Anna; Accogli, Gianluca; Cremonesi, Fausto; Desantis, Salvatore

    2014-01-01

    Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)β1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galβ1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galβ1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells.

  5. Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

    PubMed

    Lange-Consiglio, Anna; Accogli, Gianluca; Cremonesi, Fausto; Desantis, Salvatore

    2014-01-01

    Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)β1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galβ1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galβ1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells. PMID:26337136

  6. The potential role of polyamines in gill epithelial remodeling during extreme hypoosmotic challenges in the Gulf killifish, Fundulus grandis.

    PubMed

    Guan, Ying; Zhang, Guo-Xia; Zhang, Shujun; Domangue, Beau; Galvez, Fernando

    2016-01-01

    Polyamines are a family of low molecular weight organic cations produced in part by the coordinated actions of arginase II (Arg II) and ornithine decarboxylase (Odc). Although gill polyamine homeostasis is affected by acute transfer to fresh water, little is known of its function in fish osmoregulation. The current study investigated the role of polyamines in the compensatory response of hypoosmotic challenge in the euryhaline fish, Fundulus grandis. Adult F. grandis were acclimated to 5 ppt water, transferred abruptly to 5, 2, 1, 0.5 and 0.1 ppt water, and assessed for osmoregulatory function, gill morphology, and polyamine homeostasis. The plasma osmolality, Na(+) concentration, and Cl(-) concentration were only significantly reduced during exposure to salinities at or below 0.5 ppt, although these effects were transient except in the 0.1 ppt treatment. The phenotype of mitochondrion-rich cells (MRCs) shifted from a seawater-type to a freshwater-type only at salinities that also produced a plasma osmotic disturbance. Hypoosmotic exposure increased the concentrations of putrescine, spermidine, and spermine in the gill over the entire 7 day period. Exposure to 0.1 ppt water also transiently increased gill caspase-3 activity and gill mRNA levels of the immediate-early response genes, c-fos and c-myc, thus tightly associating polyamines with gill remodeling during freshwater acclimation. Furthermore, arginase II and ornithine decarboxylase mRNA levels were most highly expressed in MRCs, and these levels were further increased only in the 0.1 ppt treatment. Reduction of gill polyamine levels following administration of the Odc inhibitor, alpha-dl-difluoromethylornithine (DFMO), inhibited gill caspase-3 activity, but surprisingly reduced the magnitude of the plasma osmotic imbalance elicited by exposure to 0.1 ppt water. We used isolated opercular epithelia mounted on Ussing chambers to assess the influence of polyamines on the attenuating response of hypotonic shock

  7. Actin Cytoskeleton Regulation of Epithelial Mesenchymal Transition in Metastatic Cancer Cells

    PubMed Central

    Shankar, Jay; Nabi, Ivan R.

    2015-01-01

    Epithelial-mesenchymal transition (EMT) is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D) reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells. PMID:25756282

  8. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  9. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  10. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

    PubMed Central

    West, John D; Dorà, Natalie J; Collinson, J Martin

    2015-01-01

    In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed

  11. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

  12. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  13. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    PubMed

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  14. The innate immune function of airway epithelial cells in inflammatory lung disease

    PubMed Central

    Hiemstra, Pieter S.; McCray, Paul B.; Bals, Robert

    2016-01-01

    The airway epithelium is now considered central to the orchestration of pulmonary inflammatory and immune responses, and is also key to tissue remodelling. It acts as a first barrier in the defence against a wide range of inhaled challenges, and is critically involved in the regulation of both innate and adaptive immune responses to these challenges. Recent progress in our understanding of the developmental regulation of this tissue, the differentiation pathways, recognition of pathogens and antimicrobial responses is now exploited to help understand how epithelial cell function and dysfunction contributes to the pathogenesis of a variety of inflammatory lung diseases. In the review, advances in our knowledge of the biology of airway epithelium, as well as its role and (dys)function in asthma, COPD and cystic fibrosis, are discussed. PMID:25700381

  15. Osteogenic Differentiation of Human Amniotic Epithelial Cells and Its Application in Alveolar Defect Restoration

    PubMed Central

    Jiawen, Si; Jianjun, Zhang; Jiewen, Dai; Dedong, Yu; Hongbo, Yu; Jun, Shi; Xudong, Wang; Shen, Steve G.F.

    2014-01-01

    The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect. PMID:25368378

  16. Twist1 and Slug mediate H2AX-regulated epithelial-mesenchymal transition in breast cells.

    PubMed

    Weyemi, Urbain; Redon, Christophe E; Sethi, Taresh K; Burrell, Allison S; Jailwala, Parthav; Kasoji, Manjula; Abrams, Natalie; Merchant, Anand; Bonner, William M

    2016-09-16

    The epithelial-mesenchymal transition (EMT) is thought to be essential for cancer metastasis. While chromatin remodeling is involved in EMT, which processes contribute to this remodeling remain poorly investigated. Recently, we showed that silencing or removal of the histone variant H2A.X induced mesenchymal-like characteristics, including activation of the EMT transcription factors, Slug and Zeb1 in human colon cancer cells. Here, we provide the evidence that H2A.X loss in human non-tumorigenic breast cell line MCF10A results in a robust EMT activation, as substantiated by a genome-wide expression analysis. Cells deficient for H2A.X exhibit enhanced migration and invasion, along with an activation of a set of mesenchymal genes and a concomitant repression of epithelial genes. In the breast model, the EMT-related transcription factor Twist1 cooperates with Slug to regulate EMT upon H2A.X Loss. Of interest, H2A.X expression level tightly correlates with Twist1, and to a lesser extent with Slug in the panel of human breast cancer cell lines of the NCI-60 datasets. These new findings indicate that H2A.X is involved in the EMT processes in cells of different origins but pairing with transcription factors for EMT may be tissue specific. PMID:27315462

  17. Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration

    PubMed Central

    Saera-Vila, Alfonso; Kish, Phillip E.; Louie, Ke'ale W.; Grzegorski, Steven J.; Klionsky, Daniel J.; Kahana, Alon

    2016-01-01

    ABSTRACT Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity. PMID:27467399

  18. Dysfunctional resident lung mesenchymal stem cells contribute to pulmonary microvascular remodeling.

    PubMed

    Chow, Kelsey; Fessel, Joshua P; Kaoriihida-Stansbury; Schmidt, Eric P; Gaskill, Christa; Alvarez, Diego; Graham, Brian; Harrison, David G; Wagner, David H; Nozik-Grayck, Eva; West, James D; Klemm, Dwight J; Majka, Susan M

    2013-01-01

    Pulmonary vascular remodeling and oxidative stress are common to many adult lung diseases. However, little is known about the relevance of lung mesenchymal stem cells (MSCs) in these processes. We tested the hypothesis that dysfunctional lung MSCs directly participate in remodeling of the microcirculation. We employed a genetic model to deplete extracellular superoxide dismutase (EC-SOD) in lung MSCs coupled with lineage tracing analysis. We crossed (floxp)sod3 and mT/mG reporter mice to a strain expressing Cre recombinase under the control of the ABCG2 promoter. We demonstrated In vivo that depletion of EC-SOD in lung MSCs resulted in their contribution to microvascular remodeling in the smooth muscle actin positive layer. We further characterized lung MSCs to be multipotent vascular precursors, capable of myofibroblast, endothelial and pericyte differentiation in vitro. EC-SOD deficiency in cultured lung MSCs accelerated proliferation and apoptosis, restricted colony-forming ability, multilineage differentiation potential and promoted the transition to a contractile phenotype. Further studies correlated cell dysfunction to alterations in canonical Wnt/β-catenin signaling, which were more evident under conditions of oxidative stress. Our data establish that lung MSCs are a multipotent vascular precursor population, a population which has the capacity to participate in vascular remodeling and their function is likely regulated in part by the Wnt/β-catenin signaling pathway. These studies highlight an important role for microenviromental regulation of multipotent MSC function as well as their potential to contribute to tissue remodeling.

  19. The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse.

    PubMed Central

    Wilson, C L; Heppner, K J; Rudolph, L A; Matrisian, L M

    1995-01-01

    To explore the role of the matrix metalloproteinase matrilysin (MAT) in normal tissue remodeling, we cloned the murine homologue of MAT from postpartum uterus using RACE polymerase chain reaction and examined its pattern of expression in embryonic, neonatal, and adult mice. The murine coding sequence and the corresponding predicted protein sequence were found to be 75% and 70% identical to the human sequences, respectively, and organization of the six exons comprising the gene is similar to the human gene. Northern analysis and in situ hybridization revealed that MAT is expressed in the normal cycling, pregnant, and postpartum uterus, with levels of expression highest in the involuting uterus at early time points (6 h to 1.5 days postpartum). The mRNA was confined to epithelial cells lining the lumen and some glandular structures. High constitutive levels of MAT transcripts were also detected in the small intestine, where expression was localized to the epithelial Paneth cells at the base of the crypts. Similarly, MAT expression was found in epithelial cells of the efferent ducts, in the initial segment and cauda of the epididymis, and in an extra-hepatic branch of the bile duct. MAT transcripts were detectable only by reverse transcription-polymerase chain reaction in the colon, kidney, lung, skeletal muscle, skin, stomach, juvenile uterus, and normal, lactating, and involuting mammary gland, as was expression primarily late in embryogenesis. Analysis of MAT expression during postnatal development indicated that although MAT is expressed in the juvenile small intestine and reproductive organs, the accumulation of significant levels of MAT mRNA appears to correlate with organ maturation. These results show that MAT expression is restricted to specific organs in the mouse, where the mRNA is produced exclusively by epithelial cells, and suggest that in addition to matrix degradation and remodeling, MAT may play an important role in the differentiated function of

  20. Mechanical strain induces involution-associated events in mammary epithelial cells

    PubMed Central

    Quaglino, Ana; Salierno, Marcelo; Pellegrotti, Jesica; Rubinstein, Natalia; Kordon, Edith C

    2009-01-01

    Background Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device

  1. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

    PubMed Central

    Aoshiba, K; Nagai, A

    2003-01-01

    Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke. PMID:19570263

  2. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

    PubMed Central

    Aoshiba, K; Nagai, A

    2003-01-01

    Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke.

  3. Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells.

    PubMed

    Cho, Kyoung Bin; Cho, Min Kyong; Lee, Won Young; Kang, Keon Wook

    2010-07-28

    The c-myc gene is frequently overexpressed in human breast cancer and its target genes are involved in tumorigenesis. Epithelial mesenchymal transitions (EMT), where cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, are associated with invasion and motility of cancer cells. Basal E-cadherin expression was down-regulated in c-myc overexpressing MCF10A (c-myc-MCF10A) cells compared to GFP-overexpressing MCF10A (GFP-MCF10A) cells, while N-cadherin was distinctly increased in c-myc-MCF10A cells. Given that glycogen synthase kinase-3beta (GSK-3beta) and the snail axis have key roles in E-cadherin deregulation during EMT, we investigated the role of GSK-3beta/snail signaling pathways in the induction of EMT by c-myc overexpression. In contrast to GFP-MCF10A cells, both the transcriptional activity and the ubiquitination-dependent protein stability of snail were enhanced in c-myc-MCF10A cells, and this was reversed by GSK-3beta overexpression. We also found that c-myc overexpression inhibits GSK-3beta activity through activation of extracellular signal-regulated kinase (ERK). Inhibition of ERK by dominant negative mutant transfection or chemical inhibitor significantly suppressed snail gene transcription. These results suggest that c-myc overexpression during transformation of mammary epithelial cells (MEC) is involved in EMTs via ERK-dependent GSK-3beta inactivation and subsequent snail activation.

  4. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  5. Sheep stromal-epithelial cell interactions and ovarian tumor progression.

    PubMed

    Wang-Johanning, Feng; Huang, Miao; Liu, Jinsong; Rycaj, Kiera; Plummer, Joshua B; Barnhart, Kirstin F; Satterfield, William C; Johanning, Gary L

    2007-11-15

    Previous studies suggest that underlying ovarian stromal cues may regulate the ovarian surface epithelium. However, little is known about the interaction between ovarian stromal cells (OSC) and ovarian surface epithelial cells (OSE) under normal physiologic and pathologic conditions, largely because of the lack of a suitable model. In the current study, the OSC obtained from a sheep were immortalized with SV-40 T/t antigen (designated IOSC) and telomerase reverse transcriptase (designated IOSCH), followed by transfection with the oncogenic allele of the human H-Ras oncogene (designated IOSChR). IOSC cells transfected with H-Ras before immortalization with telomerase were designated IOSCRH. These sheep OSCs were used in both in vitro and in vivo model systems to evaluate mechanisms by which OSCs influence ovarian tumor progression. Normal sheep OSCs were found to inhibit the growth of SKOV3 and OVCAR3 human ovarian cancer cells, but not normal sheep OSE and human OSE cells (hOSE137 cells). In contrast, IOSChR and IOSCRH cells stimulated the growth of normal sheep and human OSE cells, as well as cancer cells. These findings were confirmed by in vivo studies. Our data provide compelling support for the importance of stromal-epithelial cell interactions during tumor progression, and show for the first time that immortalized and transformed OSCs promote growth of ovarian epithelial tumors.

  6. [Methotrexate as inducer of proinflammatory cytokines by epithelial cells].

    PubMed

    Morón-Medina, Alejandra; Viera, Ninoska; de Morales, Thaís Rojas; Alcocer, Sirley; Bohorquez, Dinorath

    2014-03-01

    Methotrexate (MTX), a drug commonly used in childhood cancer, has also been indicated as a cytotoxic agent of the oral mucosa, which can trigger the inflammatory process and increase the vascularity of epithelial tissues during the early stages of oral mucositis. The aim of this study was to determine the production of proinflammatory cytokines IL-1beta, IL-6 y TNF-alpha in epithelial cell cultures treated with MTX. Epithelial cells of human larynx, obtained from the cell line Hep-2, were cultured with different doses of MTX during different incubation times. The drug cytotoxicity was analyzed by means of the colorimetric test, which is based on the metabolic reduction of the bromide of 3-(4, 5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT); and the proinflammatory cytokines production by the test enzyme-linked immunosorbent assay (ELISA). Cultures of HEp-2 cells showed increased production of proinflammatory cytokines at 72 hours with 0.32 microM of MTX. These results suggest that depending on the dose and exposure time, MTX alters the physiology of human epithelial cells, which may play an important role during the phases of initiation and development of oral mucositis. PMID:24758098

  7. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  8. Myeloid-Epithelial-Reproductive Receptor Tyrosine Kinase and Milk Fat Globule Epidermal Growth Factor 8 Coordinately Improve Remodeling After Myocardial Infarction via Local Delivery of Vascular Endothelial Growth Factor

    PubMed Central

    Howangyin, Kiave-Yune; Zlatanova, Ivana; Pinto, Cristina; Ngkelo, Anta; Cochain, Clément; Rouanet, Marie; Vilar, José; Lemitre, Mathilde; Stockmann, Christian; Fleischmann, Bernd K.; Mallat, Ziad

    2016-01-01

    Background— In infarcted heart, improper clearance of dying cells by activated neighboring phagocytes may precipitate the transition to heart failure. We analyzed the coordinated role of 2 major mediators of efferocytosis, the myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and the milk fat globule epidermal growth factor (Mfge8), in directing cardiac remodeling by skewing the inflammatory response after myocardial infarction. Methods and Results— We generated double-deficient mice for Mertk and Mfge8 (Mertk−/−/Mfge8−/−) and challenged them with acute coronary ligature. Compared with wild-type, Mertk-deficient (Mertk−/−), or Mfge8-deficient (Mfge8−/−) animals, Mertk−/−/Mfge8−/− mice displayed greater alteration in cardiac function and remodeling. Mertk and Mfge8 were expressed mainly by cardiac Ly6CHigh and Low monocytes and macrophages. In parallel, Mertk−/−/Mfge8−/− bone marrow chimeras manifested increased accumulation of apoptotic cells, enhanced fibrotic area, and larger infarct size, as well as reduced angiogenesis. We found that the abrogation of efferocytosis affected neither the ability of circulating monocytes to infiltrate cardiac tissue nor the number of resident Ly6CHigh and Ly6CHow monocytes/macrophages populating the infarcted milieu. In contrast, combined Mertk and Mfge8 deficiency in Ly6CHigh/Ly6CLow monocytes/macrophages either obtained from in vitro differentiation of bone marrow cells or isolated from infarcted hearts altered their capacity of efferocytosis and subsequently blunted vascular endothelial growth factor A (VEGFA) release. Using LysMCre+/VEGFAfl/fl mice, we further identified an important role for myeloid-derived VEGFA in improving cardiac function and angiogenesis. Conclusions— After myocardial infarction, Mertk- and Mfge8-expressing monocyte/macrophages synergistically engage the clearance of injured cardiomyocytes, favoring the secretion of VEGFA to locally repair the

  9. Establishment of a rat nasal epithelial tumor cell line.

    PubMed

    Hood, A T; Currie, D; Garte, S J

    1987-04-01

    A new cell line designated NAS 2BL has been established from a rat nasal tumor induced by inhalation of the direct-acting carcinogen methylmethane sulfonate. The cells are epithelial in morphology, have a generation time of 34 h, require 10% fetal bovine serum for optimal growth, and exhibit keratinization at confluence. The karyotype is aneuploid, with several marker chromosomes, and the cells are transformed by the criterion of nude mouse tumorigenicity.

  10. Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

    PubMed

    Panneck, Alexandra Regina; Rafiq, Amir; Schütz, Burkhard; Soultanova, Aichurek; Deckmann, Klaus; Chubanov, Vladimir; Gudermann, Thomas; Weihe, Eberhard; Krasteva-Christ, Gabriela; Grau, Veronika; del Rey, Adriana; Kummer, Wolfgang

    2014-12-01

    Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

  11. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry.

  12. The skeleton in the closet: actin cytoskeletal remodeling in β-cell function.

    PubMed

    Arous, Caroline; Halban, Philippe A

    2015-10-01

    Over the last few decades, biomedical research has considered not only the function of single cells but also the importance of the physical environment within a whole tissue, including cell-cell and cell-extracellular matrix interactions. Cytoskeleton organization and focal adhesions are crucial sensors for cells that enable them to rapidly communicate with the physical extracellular environment in response to extracellular stimuli, ensuring proper function and adaptation. The involvement of the microtubular-microfilamentous cytoskeleton in secretion mechanisms was proposed almost 50 years ago, since when the evolution of ever more sensitive and sophisticated methods in microscopy and in cell and molecular biology have led us to become aware of the importance of cytoskeleton remodeling for cell shape regulation and its crucial link with signaling pathways leading to β-cell function. Emerging evidence suggests that dysfunction of cytoskeletal components or extracellular matrix modification influences a number of disorders through potential actin cytoskeleton disruption that could be involved in the initiation of multiple cellular functions. Perturbation of β-cell actin cytoskeleton remodeling could arise secondarily to islet inflammation and fibrosis, possibly accounting in part for impaired β-cell function in type 2 diabetes. This review focuses on the role of actin remodeling in insulin secretion mechanisms and its close relationship with focal adhesions and myosin II.

  13. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  14. Lung epithelial cell death induced by oil-dispersant mixtures.

    PubMed

    Wang, He; Shi, Yongli; Major, Danielle; Yang, Zhanjun

    2012-08-01

    The dispersants used in oil spill disasters are claimed to be safe, but increased solubility of high-molecular-weight components in crude oil is of public health concern. The water-accommodated fractions (WAF) of crude oil mixed with dispersants may become airborne and cause lung epithelial damage when inhaled. This study was designed to examine the cell death and related death pathways of lung epithelial cells in response to WAF. Cultured A549 cells were treated for 2 or 24h with different concentrations of WAF. The WAF was prepared by mixing each of the dispersants (Corexit EC9527A, Corexit EC9500A and Corexit EC9580A) with crude oil for extraction with PBS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay, lactate dehydrogenase assay, morphology and cleaved caspase 9 protein, and microtubule-associated protein 1 light chain 3 were all used to measure cell viability, necrosis, apoptosis and autophagy quantitation, respectively. Results showed that the WAF of oil-dispersant mixtures caused cell death in the lung epithelial cells, in a dose-dependent manner, with the major cellular pathways of necrosis and apoptosis involved. Autophagy also occurred in cells exposed to WAF mixtures at lower concentrations before any detectable cell death, indicating greater sensitivity to WAF exposure. The three types of cell behavior, namely necrosis, apoptosis and autophagy, may play different roles in oil spill-related respiratory disorders. PMID:22504303

  15. Direct Role for Smooth Muscle Cell Mineralocorticoid Receptors in Vascular Remodeling: Novel Mechanisms and Clinical Implications

    PubMed Central

    Koenig, Jenny B.; Jaffe, Iris Z.

    2014-01-01

    The mineralocorticoid receptor (MR) is a key regulator of blood pressure. MR-antagonist drugs are used to treat hypertension and heart failure, resulting in decreased mortality by mechanisms that are not completely understood. In addition to the kidney, MR is also expressed in the smooth muscle cells (SMCs) of the vasculature, where it is activated by the hormone aldosterone and affects the expression of genes involved in vascular function at the cellular and systemic levels. Following vascular injury due to mechanical or physiological stresses, vessels undergo remodeling resulting in SMC hypertrophy, migration, and proliferation, as well as vessel fibrosis. Exuberant vascular remodeling is associated with poor outcomes in cardiovascular patients. This review compiles recent findings on the specific role of SMC-MR in the vascular remodeling process. The development and characterization of a SMC-specific MR-knockout mouse has demonstrated a direct role for SMC-MR in vascular remodeling. Additionally, several novel mechanisms contributing to SMC-MR-mediated vascular remodeling have been identified and are reviewed here, including Rho-kinase signaling, placental growth factor signaling through vascular endothelial growth factor type 1 receptor, and galectin signaling. PMID:24633842

  16. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    PubMed

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  17. In vitro differentiation of a cloned bovine mammary epithelial cell.

    PubMed

    Rose, Michael T; Aso, Hisashi; Yonekura, Shinichi; Komatsu, Tokushi; Hagino, Akihiko; Ozutsumi, Kyouhei; Obara, Yoshiaki

    2002-08-01

    The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of alpha-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced alpha-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce alpha-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

  18. Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

    PubMed Central

    Centeno, A; Davis, C P; Cohen, M S; Warren, M M

    1983-01-01

    The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Images PMID:6132878

  19. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  20. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  1. Chronic Alcohol Exposure Renders Epithelial Cells Vulnerable to Bacterial Infection

    PubMed Central

    Wood, Stephen; Pithadia, Ravi; Rehman, Tooba; Zhang, Lijuan; Plichta, Jennifer; Radek, Katherine A.; Forsyth, Christopher; Keshavarzian, Ali; Shafikhani, Sasha H.

    2013-01-01

    Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier’s capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol’s deleterious effects on cells may be reversible. PMID:23358457

  2. Chronic alcohol exposure renders epithelial cells vulnerable to bacterial infection.

    PubMed

    Wood, Stephen; Pithadia, Ravi; Rehman, Tooba; Zhang, Lijuan; Plichta, Jennifer; Radek, Katherine A; Forsyth, Christopher; Keshavarzian, Ali; Shafikhani, Sasha H

    2013-01-01

    Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier's capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol's deleterious effects on cells may be reversible. PMID:23358457

  3. Marked induction of matrix metalloproteinase-10 by respiratory syncytial virus infection in human nasal epithelial cells.

    PubMed

    Hirakawa, Satoshi; Kojima, Takashi; Obata, Kazufumi; Okabayashi, Tamaki; Yokota, Shin-Ichi; Nomura, Kazuaki; Obonai, Toshimasa; Fuchimoto, Jun; Himi, Tetsuo; Tsutsumi, Hiroyuki; Sawada, Norimasa

    2013-12-01

    Respiratory syncytial virus (RSV) is an important pathogen of bronchiolitis, asthma, and severe lower respiratory tract disease in infants and young children. Matrix metalloproteinases (MMPs) play key roles in viral infection, inflammation and remodeling of the airway. However, the roles and regulation of MMPs in human nasal epithelial cells (HNECs) after RSV infection remain unclear. To investigate the regulation of MMP induced after RSV infection in HNECs, an RSV-infected model of HNECs in vitro was used. It was found that mRNA of MMP-10 was markedly increased in HNECs after RSV infection, together with induction of mRNAs of MMP-1, -7, -9, and -19. The amount of MMP-10 released from HNECs was also increased in a time-dependent manner after RSV infection as was that of chemokine RANTES. The upregulation of MMP-10 in HNECs after RSV infection was prevented by inhibitors of NF-κB and pan-PKC with inhibition of RSV replication, whereas it was prevented by inhibitors of JAK/STAT, MAPK, and EGF receptors without inhibition of RSV replication. In lung tissue of an infant with severe RSV infection in which a few RSV antibody-positive macrophages were observed, MMP-10 was expressed at the apical side of the bronchial epithelial cells and alveolar epithelial cells. In conclusion, MMP-10 induced by RSV infection in HNECs is regulated via distinct signal transduction pathways with or without relation to RSV replication. MMP-10 may play an important role in the pathogenesis of RSV diseases and it has the potential to be a novel marker and therapeutic target for RSV infection.

  4. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  5. Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination

    PubMed Central

    Le, Caroline P.; Nowell, Cameron J.; Kim-Fuchs, Corina; Botteri, Edoardo; Hiller, Jonathan G.; Ismail, Hilmy; Pimentel, Matthew A.; Chai, Ming G.; Karnezis, Tara; Rotmensz, Nicole; Renne, Giuseppe; Gandini, Sara; Pouton, Colin W.; Ferrari, Davide; Möller, Andreas; Stacker, Steven A.; Sloan, Erica K.

    2016-01-01

    Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes. PMID:26925549

  6. Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

    PubMed Central

    Thevenot, Paul T.; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D.; Chustz, Regina E.; Mahne, Sarah; Kelley, Matthew A.; Hebert, Valeria Y.; Dellinger, Barry; Dugas, Tammy R.; DeMayo, Francesco J.

    2013-01-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  7. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  8. miR-124 regulates fetal pulmonary epithelial cell maturation

    PubMed Central

    Wang, Yang; Huang, Chaoqun; Chintagari, Narendranath Reddy; Xi, Dong; Weng, Tingting

    2015-01-01

    MicroRNAs are a family of small noncoding RNAs that regulate the expression of their target proteins at the posttranscriptional level. Their functions cover almost every aspect of cell physiology. However, the roles of microRNAs in fetal lung development are not completely understood. The objective of this study is to investigate the regulation and molecular mechanisms of alveolar epithelial cell maturation during fetal lung development by miR-124. We discovered that miR-124 was downregulated during rat fetal lung development and predominantly expressed in the epithelial cells at late stage of the lung development. Overexpression of miR-124 with an adenovirus vector led to the inhibition of epithelial maturation in rat fetal lung organ cultures and fetal alveolar epithelial type II cells, as demonstrated by a decrease in the type II cell marker expression and an increase in glycogen content. We further demonstrated by luciferase reporter assays that miR-124 inhibited the NF-κB, cAMP/PKA, and MAPK/ERK pathways. In addition, nuclear factor I/B (NFIB), a critical protein in fetal lung maturation, was validated as a direct target of miR-124. Furthermore, miR-124 expression was induced by the Wnt/β-catenin signaling pathway through a direct interaction of LEF1 and the miR-124 promoter region. We concluded that miR-124 downregulation is critical to fetal lung epithelial maturation and miR-124 inhibits this maturation process at least partially through the inhibition of NFIB. PMID:26071557

  9. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  10. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells. PMID:27398446

  11. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  12. Notch Signaling in Meibomian Gland Epithelial Cell Differentiation

    PubMed Central

    Gidfar, Sanaz; Afsharkhamseh, Neda; Sanjari, Sara; Djalilian, Ali R.

    2016-01-01

    Purpose Notch1 was previously shown to play a critical role in murine meibomian gland function and maintenance. In this study, we have examined the expression and activation of Notch pathway in human meibomian gland epithelial cells in vitro. Methods An immortalized human meibomian gland epithelial cell (HMGEC) line was cultured under proliferative and differentiative conditions. Expression of Notch receptors and ligands were evaluated by quantitative PCR and Western blot. The effect of Notch inhibition and induction on oil production was also assessed. Results Human meibomian gland epithelial cell expressed Notch1, Notch2, Notch3, Jagged1, Jagged2, Delta-like 1, and Delta-like 3. The level of cleaved (activated) Notch1 strongly increased with differentiation. The expression of Notch3 was inversely correlated with proliferation. Induction and inhibition of Notch1 led to an increase and decrease in the amount of oil production, respectively. Conclusions Notch signaling appears to play an important role in human meibomian gland epithelial differentiation and oil production. This may provide a potential therapeutic pathway for treating meibomian gland dysfunction. PMID:26943148

  13. Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration

    PubMed Central

    Dudakov, Jarrod A.; Jenq, Robert R.; Velardi, Enrico; Young, Lauren F.; Smith, Odette M.; Lawrence, Gillian; Ivanov, Juliet A.; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L.; O'Rourke, Kevin P.; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas; Nieuwenhuis, Edward E.; Shroyer, Noah F.; Liu, Chen; Kolesnick, Richard

    2015-01-01

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  14. Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

    PubMed

    Svirshchevskaya, E V; Poltavtseva, R A; Beletskii, I P; Selezneva, I I; Sukhikh, G T

    2016-08-01

    We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin. PMID:27590756

  15. Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

    PubMed Central

    Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

    2015-01-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

  16. COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

    EPA Science Inventory

    Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

  17. Midbody remnant licenses primary cilia formation in epithelial cells.

    PubMed

    Ott, Carolyn M

    2016-08-01

    Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model. PMID:27482049

  18. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    SciTech Connect

    Doupnik, C.A.; Leikauf, G.D. )

    1990-10-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with (3H)arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. (3H)arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein.

  19. Review: corneal epithelial stem cells, their niche and wound healing.

    PubMed

    Castro-Muñozledo, Federico

    2013-01-01

    Stem cells emerged as a concept during the second half of 19(th) century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet's membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  20. Review: Corneal epithelial stem cells, their niche and wound healing

    PubMed Central

    2013-01-01

    Stem cells emerged as a concept during the second half of 19th century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet’s membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  1. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

    PubMed

    Gomperts, Brigitte N; Belperio, John A; Rao, P Nagesh; Randell, Scott H; Fishbein, Michael C; Burdick, Marie D; Strieter, Robert M

    2006-02-01

    Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases. PMID:16424223

  2. Aldosterone promotes vascular remodeling by direct effects on smooth muscle cell mineralocorticoid receptors

    PubMed Central

    Pruthi, Dafina; McCurley, Amy; Aronovitz, Mark; Galayda, Carol; Karumanchi, S. Ananth; Jaffe, Iris Z.

    2014-01-01

    Objective Vascular remodeling occurs after endothelial injury resulting in smooth muscle cell (SMC) proliferation and vascular fibrosis. We previously demonstrated that the blood pressure-regulating hormone aldosterone enhances vascular remodeling in mice at sites of endothelial injury in a placental growth factor (PlGF)-dependent manner. We now test the hypothesis that SMC mineralocorticoid receptors (MR) directly mediate the remodeling effects of aldosterone and further explore the mechanism. Approach and Results A wire-induced carotid injury model was performed in wild type (WT) mice and mice with inducible SMC-specific deletion of MR (SMC-MR-KO). Aldosterone did not affect re-endothelialization after injury in WT mice. Deletion of SMC-MR prevented the 79% increase in SMC proliferation induced by aldosterone after injury in MR-Intact littermates. Moreover, both injury-induced and aldosterone-enhanced vascular fibrosis were attenuated in SMC-MR-KO mice. Further exploration of the mechanism revealed that aldosterone-induced vascular remodeling is prevented by blockade of the PlGF-specific receptor, VEGFR1, in vivo. Immunohistochemistry of carotid vessels shows that the induction of VEGFR1 expression in SMC after vascular injury is attenuated by 72% in SMC-MR-KO mice. Moreover, aldosterone induction of vascular PlGF mRNA expression and protein release are also prevented in vessels lacking SMC-MR. Conclusions These studies reveal that SMC-MR is necessary for aldosterone-induced vascular remodeling independent of renal effects on blood pressure. SMC-MR contributes to induction of SMC VEGFR1 in the area of vascular injury and to aldosterone-enhanced vascular PlGF expression and hence the detrimental effects of aldosterone are prevented by VEGFR1-blockade. This study supports exploring MR antagonists and VEGFR1-blockade to prevent pathological vascular remodeling induced by aldosterone. PMID:24311380

  3. C/EBPα and the Vitamin D Receptor Cooperate in the Regulation of Cathelicidin in Lung Epithelial Cells.

    PubMed

    Dhawan, Puneet; Wei, Ran; Sun, Cheng; Gombart, Adrian F; Koeffler, H Phillip; Diamond, Gill; Christakos, Sylvia

    2015-02-01

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and the vitamin D receptor (VDR) have been reported to have an important role in the regulation of innate immunity. We earlier reported that the antimicrobial peptide cathelicidin is induced by 1,25(OH)2D3 in normal human bronchial epithelial cells with a resultant increase in antimicrobial activity against airway pathogens. In this study, we demonstrate that C/EBP alpha (C/EBPα) is a potent enhancer of human cathelicidin antimicrobial peptide (CAMP) gene transcription in human lung epithelial cells. In addition we found that C/EBPα functionally cooperates with VDR in the regulation of CAMP transcription. A C/EBP binding site was identified at -627/-619 within the CAMP promoter, adjacent to the vitamin D response element (VDRE; -615/-600). Mutation of this site markedly attenuated the transcriptional response to C/EBPα as well as to 1,25(OH)2D3, further indicating cooperation between these two factors in the regulation of CAMP. ChIP analysis using 1,25(OH)2D3 treated human lung epithelial cells showed C/EBPα and VDR binding to the CAMP promoter. C/EBPα has previously been reported to cooperate with Brahma (Brm), an ATPase that is component of the SWI/SNF chromatin remodeling complex. We found that dominant negative Brm significantly inhibited C/EBPα as well as 1,25(OH)2D3 mediated induction of CAMP transcription, suggesting the functional involvement of Brm. These findings define novel mechanisms involving C/EBPα, SWI/SNF, and 1,25(OH)2D3 in the regulation of CAMP in lung epithelial cells. These mechanisms of enhanced activation of the CAMP gene in lung epithelial cells suggest potential candidates for the development of modulators of innate immune responses for adjunct therapy in the treatment of airway infections. PMID:25078430

  4. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; hafiz, Khandakar musabbir bin; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  5. Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells.

    PubMed

    Chanoux, Rebecca A; Shubin, Calla B; Robay, Amal; Suaud, Laurence; Rubenstein, Ronald C

    2013-10-01

    The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface. PMID:23885065

  6. Alveolar epithelial type II cell: defender of the alveolus revisited

    PubMed Central

    Fehrenbach, Heinz

    2001-01-01

    In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2) cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today. PMID:11686863

  7. Host epithelial geometry regulates breast cancer cell invasiveness

    PubMed Central

    Boghaert, Eline; Gleghorn, Jason P.; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment. PMID:23150585

  8. DNA analysis of epithelial cell suspensions

    SciTech Connect

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  9. To grow mouse mammary epithelial cells in culture

    PubMed Central

    1984-01-01

    Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures. PMID:6699079

  10. Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture.

    PubMed

    Bennett, M R

    1999-02-01

    Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as an important process in a variety of human vascular diseases, including atherosclerosis, arterial injury, and restenosis after angioplasty. VSMC apoptosis is regulated by interactions between the local cell-cell and cytokine environment within the arterial wall, and the expression of pro- and anti-apoptotic proteins by the cell, including death receptors, proto-oncogenes and tumour suppressor genes. This review summarises our current knowledge of the occurrence and mechanisms underlying VSMC apoptosis in atherosclerosis and arterial remodelling.

  11. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  12. Helicobacter pylori increases proliferation of gastric epithelial cells.

    PubMed

    Fan, X G; Kelleher, D; Fan, X J; Xia, H X; Keeling, P W

    1996-01-01

    The direct and indirect effects of helicobacter pylori on cell kinetics of gastric epithelial cell line AGS were investigated by flow cytometric analysis of Ki-67 positive cells and by MTT assay. Flow cytometric analysis of Ki-67 positivity permits detection of cells that are in S-phase, whereas the MTT assay is a colometric measure of the number of viable cells. In the absence of added stimulants, 23.06 (4.88)% mean (SD) of AGS cells were Ki-67 positive. When cells were preincubated in the presence of H pylori, there was a significant increase in Ki-67 positivity (66.20 (7.89)%, p < 0.001). This increase was not seen in cells cultured in the presence of Campylobacter jejuni (24.63 (8.11)% or Escherichia coli (21.66 (9.78)%). Pre-incubation of AGS cells with supernatants from both H pylori and mitogen activated peripheral blood lymphocytes also increased the per cent of cells that were Ki-67 positive (72.93 (8.68) and 69.96 (12.35)%; p, 0.001) respectively. Similar results were also found in MTT assay. These data show that both H pylori directly and the immune/inflammatory response to H pylori indirectly can influence the rate of epithelial cell proliferation, suggesting this bacterium may be an initiating step in gastric carcinogenesis and an important co-carcinogenic factor in H pylori positive subjects.

  13. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  14. Spatiotemporal regulation of chemical reaction kinetics of cell surface molecules by active remodeling of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Mayor, Satyajit; Rao, Madan

    2010-03-01

    Cell surface proteins such as lipid tethered GPI-anchored proteins and Ras-proteins are distributed as monomers and nanoclusters on the surface of living cells. Recent work from our laboratory suggests that the spatial distribution and dynamics of formation and breakup of these nanoclusters is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we propose a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. Here we study the consequences of such active actin based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that active remodeling of cortical actin, can give rise to a dramatic increase in the reaction efficiency and output levels. In general, such actin driven clustering of membrane proteins could be a cellular mechanism to spatiotemporally regulate and amplify local chemical reaction rates, in the context of signalling and endocytosis.

  15. Clostridium difficile toxin A binding to human intestinal epithelial cells.

    PubMed

    Smith, J A; Cooke, D L; Hyde, S; Borriello, S P; Long, R G

    1997-11-01

    Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.

  16. miR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings.

    PubMed

    Bolin, Kelsey; Rachmaninoff, Nicholas; Moncada, Kea; Pula, Katharine; Kennell, Jennifer; Buttitta, Laura

    2016-04-01

    The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling - pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate. PMID:26902111

  17. Stiffness nanotomography of human epithelial cancer cells

    NASA Astrophysics Data System (ADS)

    Staunton, Jack R.; Doss, Bryant L.; Gilbert, C. Michael; Kasas, Sandor; Ros, Robert

    2012-02-01

    The mechanical stiffness of individual cells is important in both cancer initiation and metastasis. We present atomic force microscopy (AFM) based nanoindentation experiments on various human mammary and esophagus cell lines covering the spectrum from normal immortalized cells to highly metastatic ones. The combination of an AFM with a confocal fluorescence lifetime imaging microscope (FLIM) in conjunction with the ability to move the sample and objective independently allow for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. This enables us to correlate the mechanical properties with the point of indentation in the FLIM image. We are using force-volume measurements as well as force indentation curves on distinct points on the cells to compare the elastic moduli of the nuclei, nucleoli, and the cytoplasm, and how they vary within and between individual cells and cell lines. Further, a detailed analysis of the force-indentation curves allows study of the cells' mechanical properties at different indentation depths and to generate 3D elasticity maps.

  18. Axl modulates immune activation of smooth muscle cells in vein graft remodeling.

    PubMed

    Batchu, Sri N; Xia, Jixiang; Ko, Kyung Ae; Doyley, Marvin M; Abe, Jun-Ichi; Morrell, Craig N; Korshunov, Vyacheslav A

    2015-09-15

    The pathophysiological mechanisms of the immune activation of smooth muscle cells are not well understood. Increased expression of Axl, a receptor tyrosine kinase, was recently found in arteries from patients after coronary bypass grafts. In the present study, we hypothesized that Axl-dependent immune activation of smooth muscle cells regulates vein graft remodeling. We observed a twofold decrease in intimal thickening after vascular and systemic depletion of Axl in vein grafts. Local depletion of Axl had the greatest effect on immune activation, whereas systemic deletion of Axl reduced intima due to an increase in apoptosis in vein grafts. Primary smooth muscle cells isolated from Axl knockout mice had reduced proinflammatory responses by prevention of the STAT1 pathway. The absence of Axl increased suppressor of cytokine signaling (SOCS)1 expression in smooth muscle cells, a major inhibitory protein for STAT1. Ultrasound imaging suggested that vascular depletion of Axl reduced vein graft stiffness. Axl expression determined the STAT1-SOCS1 balance in vein graft intima and progression of the remodeling. The results of this investigation demonstrate that Axl promotes STAT1 signaling via inhibition of SOCS1 in activated smooth muscle cells in vein graft remodeling.

  19. Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum.

    PubMed

    Thiriet, C; Hayes, J J

    1999-03-01

    Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed. PMID:10219572

  20. The Roles of SNF2/SWI2 Nucleosome Remodeling Enzymes in Blood Cell Differentiation and Leukemia

    PubMed Central

    Prasad, Punit; Lennartsson, Andreas; Ekwall, Karl

    2015-01-01

    Here, we review the role of sucrose nonfermenting (SNF2) family enzymes in blood cell development. The SNF2 family comprises helicase-like ATPases, originally discovered in yeast, that can remodel chromatin by changing chromatin structure and composition. The human genome encodes 30 different SNF2 enzymes. SNF2 family enzymes are often part of multisubunit chromatin remodeling complexes (CRCs), which consist of noncatalytic/auxiliary subunit along with the ATPase subunit. However, blood cells express a limited set of SNF2 ATPases that are necessary to maintain the pool of hematopoietic stem cells (HSCs) and drive normal blood cell development and differentiation. The composition of CRCs can be altered by the association of specific auxiliary subunits. Several auxiliary CRC subunits have specific functions in hematopoiesis. Aberrant expressions of SNF2 ATPases and/or auxiliary CRC subunit(s) are often observed in hematological malignancies. Using large-scale data from the International Cancer Genome Consortium (ICGC) we observed frequent mutations in genes encoding SNF2 helicase-like enzymes and auxiliary CRC subunits in leukemia. Hence, orderly function of SNF2 family enzymes is crucial for the execution of normal blood cell developmental program, and defects in chromatin remodeling caused by mutations or aberrant expression of these proteins may contribute to leukemogenesis. PMID:25789315

  1. Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

    PubMed Central

    Lee, Wing-Kee; Dittmar, Thomas

    2014-01-01

    A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin. PMID:25407650

  2. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  3. Vimentin contributes to human mammary epithelial cell migration.

    PubMed

    Gilles, C; Polette, M; Zahm, J M; Tournier, J M; Volders, L; Foidart, J M; Birembaut, P

    1999-12-01

    Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

  4. Remodeling of cytoskeletal architecture of nonneuronal cells induced by synapsin.

    PubMed Central

    Han, H Q; Greengard, P

    1994-01-01

    The synapsins, a family of neuron-specific phosphoproteins, have been implicated in the functional and structural maturation of synapses. The cell biological basis for these effects is unknown. In vitro, the synapsins interact with cytoskeletal elements including actin. To examine, in vivo, the possible effect of the synapsins on cytoskeletal organization and cell morphology, we have transfected each of the four known members of the synapsin family into nonneuronal cells. We report here that synapsin expression in fibroblast cells gives rise to an alteration in cell morphology that is associated with formation of highly elongated processes. This morphological change is accompanied by a reorganization of filamentous actin (F-actin) characterized by disruption of existing stress fibers and formation of bundles of actin cables in the elongated processes. These results suggest that interactions of the synapsins with actin, and possibly with other cytoskeletal elements, may play a role in the morphological differentiation of neurons. Images PMID:8078922

  5. Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells.

    PubMed

    Chen, Ying-Ting; Li, Wei; Hayashida, Yasutaka; He, Hua; Chen, Szu-Yu; Tseng, David Y; Kheirkhah, Ahmad; Tseng, Scheffer C G

    2007-08-01

    Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.

  6. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  7. Vitamin C inhibit the proliferation, migration and epithelial-mesenchymal-transition of lens epithelial cells by destabilizing HIF-1α

    PubMed Central

    Zhao, Lin; Quan, Yanlong; Wang, Jianming; Wang, Feng; Zheng, Yuping; Zhou, Aiyi

    2015-01-01

    Posterior capsular opacification (PCO), the main complication of cataract surgery, is mainly caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the residual lens epithelial cells (LECs).Vitamin C was reported to reduce the risk of forming a cataract. However, there has been no study showing the association between vitamin C and PCO. In this study, we found that vitamin C could inhibit the migration and proliferation of human lens epithelial cells. We also found that vitamin C could increase the proline hydroxylation of HIF-1α and reduce the activity of HIF-1α. Moreover, vitamin C could not inhibit the activity of proline-mutant HIF-1α (402/564). Overexpression of wild-type HIF-1α or proline-mutant HIF-1α was found to increase the proliferation and migration of human lens epithelial cells. Differently, vitamin C could inhibit the proliferation and migration in wild-type HIF-1α-overexpressing lens epithelial cells but not the proline-mutant HIF-1α-overexpressing cells. Additionally, vitamin C was also found to inhibit the expression of EMT transcription factors TWIST. We then found that vitamin C could repress the EMT phenotypes induced by the overexpression of wild-type HIF-1α but not the proline-mutant HIF-1α. These results provide evidence that vitamin C plays a role in the repression of proliferation, migration, and EMT of human lens epithelial cells by destabilizing HIF-1α. PMID:26628999

  8. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

    PubMed

    Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

    2015-11-01

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.

  9. Proteomic profiling of rat lung epithelial cells induced by acrolein

    PubMed Central

    Sarkar, Poonam; Hayes, Barbara E.

    2009-01-01

    Aims Acrolein is a highly toxic unsaturated aldehyde and is also an endogenous byproduct produced from lipid peroxidation. It can be formed from the breakdown of certain pollutants in outdoor air or from burning tobacco or gasoline. Inhalation and dermal exposure to acrolein are extremely toxic to human tissue. Although it is known that acrolein is toxic to lung tissue, no studies have attempted to address the changes induced by acrolein on a global scale. Main methods In the present study we have attempted to address the changes in global protein expression induced by acrolein using proteomics analysis in rat lung epithelial cells. Key findings Our analysis reveals a comprehensive profiling of the proteins that includes a heterogeneous class of proteins and this compels one to consider that the toxic response to acrolein is very complex. There were 34 proteins that showed changes between the control cells and after acrolein treatment. The expression of 18 proteins was increased and the expression of 16 proteins was decreased following exposure to acrolein. We have further validated two differentially expressed proteins namely annexin II (ANXII) and prohibitin (PHB) in lung epithelial cells treated with acrolein. Significance Based on the results of the overall proteomic analysis, acrolein appears to induce changes in a diverse range of proteins suggesting a complex mechanism of acrolein-induced toxicity in lung epithelial cells. PMID:19490921

  10. Serratia marcescens is injurious to intestinal epithelial cells.

    PubMed

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  11. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue.

  12. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  13. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  14. Antagonism of Stem Cell Factor/c-kit Signaling Attenuates Neonatal Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

    PubMed Central

    Young, Karen C; Torres, Eneida; Hehre, Dorothy; Wu, Shu; Suguihara, Cleide; Hare, Joshua M.

    2015-01-01

    Background Accumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis. Methods Neonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated. Results As compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation. Conclusion SCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH. PMID:26705118

  15. Dynamin-mediated endocytosis is required for tube closure, cell intercalation, and biased apical expansion during epithelial tubulogenesis in the Drosophila ovary.

    PubMed

    Peters, Nathaniel C; Berg, Celeste A

    2016-01-01

    Most metazoans are able to grow beyond a few hundred cells and to support differentiated tissues because they elaborate multicellular, epithelial tubes that are indispensable for nutrient and gas exchange. To identify and characterize the cellular behaviors and molecular mechanisms required for the morphogenesis of epithelial tubes (i.e., tubulogenesis), we have turned to the D. melanogaster ovary. Here, epithelia surrounding the developing egg chambers first pattern, then form and extend a set of simple, paired, epithelial tubes, the dorsal appendage (DA) tubes, and they create these structures in the absence of cell division or cell death. This genetically tractable system lets us assess the relative contributions that coordinated changes in cell shape, adhesion, orientation, and migration make to basic epithelial tubulogenesis. We find that Dynamin, a conserved regulator of endocytosis and the cytoskeleton, serves a key role in DA tubulogenesis. We demonstrate that Dynamin is required for distinct aspects of DA tubulogenesis: DA-tube closure, DA-tube-cell intercalation, and biased apical-luminal cell expansion. We provide evidence that Dynamin promotes these processes by facilitating endocytosis of cell-cell and cell-matrix adhesion complexes, and we find that precise levels and sub-cellular distribution of E-Cadherin and specific Integrin subunits impact DA tubulogenesis. Thus, our studies identify novel morphogenetic roles (i.e., tube closure and biased apical expansion), and expand upon established roles (i.e., cell intercalation and adhesion remodeling), for Dynamin in tubulogenesis.

  16. Oral epithelial cell responses to multispecies microbial biofilms.

    PubMed

    Peyyala, R; Kirakodu, S S; Novak, K F; Ebersole, J L

    2013-03-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms.

  17. Measles virus breaks through epithelial cell barriers to achieve transmission

    PubMed Central

    Takeda, Makoto

    2008-01-01

    Measles is a highly contagious disease that causes immunosuppression in patients. Measles virus infection has been thought to begin in the respiratory epithelium and then spread to lymphoid tissue. In this issue of the JCI, Leonard et al. provide data to suggest an alternative model of measles virus pathogenesis (see the related article beginning on page 2448). In human primary epithelial cells and rhesus monkeys in vivo, the authors show that initial infection of respiratory epithelium is not necessary for the virus to enter the host but that viral entry into epithelial cells via interaction of the virus with a receptor located on the basolateral side of the epithelium is required for viral shedding into the airway and subsequent transmission. PMID:18568081

  18. Epithelial Cell Polarity Determinant CRB3 in Cancer Development

    PubMed Central

    Li, Pingping; Mao, Xiaona; Ren, Yu; Liu, Peijun

    2015-01-01

    Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development. PMID:25552927

  19. Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation.

    PubMed

    Kim, Min Hwan; Kim, Jongshin; Hong, Hyowon; Lee, Si-Hyung; Lee, June-Koo; Jung, Eunji; Kim, Joon

    2016-03-01

    The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/TAZ-dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to the BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/TAZ. Knockdown of YAP/TAZ reduces the viability of resistant melanoma cells, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and actomyosin tension in melanoma cells suppresses both YAP/TAZ activation and PLX4032 resistance. Our siRNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/TAZ-dependent resistance pathway. These results suggest that inhibition of actin remodeling is a potential strategy to suppress resistance in BRAF inhibitor therapies.

  20. Ivermectin inhibits growth of Chlamydia trachomatis in epithelial cells.

    PubMed

    Pettengill, Matthew A; Lam, Verissa W; Ollawa, Ikechukwu; Marques-da-Silva, Camila; Ojcius, David M

    2012-01-01

    Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia. PMID:23119027

  1. Characterization of foamy epithelial surface cells in the canine endometrium.

    PubMed

    Bartel, C; Tichy, A; Walter, I

    2014-06-01

    In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, β-catenin and E-cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E-cadherin and β-catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid-accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.

  2. Role of medullary progenitor cells in epithelial cell migration and proliferation

    PubMed Central

    Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

    2014-01-01

    This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

  3. USP22 promotes epithelial-mesenchymal transition via the FAK pathway in pancreatic cancer cells.

    PubMed

    Ning, Zhen; Wang, Aman; Liang, Jinxiao; Xie, Yunpeng; Liu, Jiwei; Yan, Qiu; Wang, Zhongyu

    2014-10-01

    Epithelial-mesenchymal transition (EMT) contributes to the occurrence and development of tumors, particularly to the promotion of tumor invasion and metastasis. As a newly discovered ubiquitin hydrolase family member, USP22 plays a key role in the malignant transformation of tumors and the regulation of the cell cycle. However, recent studies on USP22 have primarily focused on its role in cell cycle regulation, and the potential mechanism underlying the promotion of tumor invasion and metastasis by abnormal USP22 expression has not been reported. Our studies revealed that the overexpression of USP22 in PANC-1 cells promoted Ezrin redistribution and phosphorylation and cytoskeletal remodeling, upregulated expression of the transcription factors Snail and ZEB1 to promote EMT, and increased cellular invasion and migration. In contrast, blockade of USP22 expression resulted in the opposite effects. In addition, the focal adhesion kinase (FAK) signaling pathway was shown to play a key role in the process of EMT induction in PANC-1 cells by USP22. Thus, the present study suggests that USP22 acts as a regulatory protein for EMT in pancreatic cancer, which may provide a new approach for the targeted therapy of pancreatic cancer. PMID:25070659

  4. Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells

    PubMed Central

    Kluz, Thomas; Cohen, Lisa; Shen, Steven S.; Costa, Max

    2016-01-01

    Cadmium is a carcinogenic metal, the mechanisms of which are not fully understood. In this study, human bronchial epithelial cells were transformed with sub-toxic doses of cadmium (0.01, 0.05, and 0.1 μM) and transformed clones were characterized for gene expression changes using RNA-seq, as well as other molecular measurements. 440 genes were upregulated and 47 genes were downregulated in cadmium clones relative to control clones over 1.25-fold. Upregulated genes were associated mostly with gene ontology terms related to embryonic development, immune response, and cell movement, while downregulated genes were associated with RNA metabolism and regulation of transcription. Several embryonic genes were upregulated, including the transcription regulator SATB2. SATB2 is critical for normal skeletal development and has roles in gene expression regulation and chromatin remodeling. Small hairpin RNA knockdown of SATB2 significantly inhibited growth in soft agar, indicating its potential as a driver of metal-induced carcinogenesis. An increase in oxidative stress and autophagy was observed in cadmium clones. In addition, the DNA repair protein O6-methylguanine-DNA-methyltransferase was depleted by transformation with cadmium. MGMT loss caused significant decrease in cell viability after treatment with the alkylating agent temozolomide, demonstrating diminished capacity to repair such damage. Results reveal various mechanisms of cadmium-induced malignant transformation in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress. PMID:27186882

  5. TCDD alters medial epithelial cell differentiation during palatogenesis

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1989-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of (3H)TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation.

  6. Connecting mechanics and bone cell activities in the bone remodeling process: an integrated finite element modeling.

    PubMed

    Hambli, Ridha

    2014-01-01

    Bone adaptation occurs as a response to external loadings and involves bone resorption by osteoclasts followed by the formation of new bone by osteoblasts. It is directly triggered by the transduction phase by osteocytes embedded within the bone matrix. The bone remodeling process is governed by the interactions between osteoblasts and osteoclasts through the expression of several autocrine and paracrine factors that control bone cell populations and their relative rate of differentiation and proliferation. A review of the literature shows that despite the progress in bone remodeling simulation using the finite element (FE) method, there is still a lack of predictive models that explicitly consider the interaction between osteoblasts and osteoclasts combined with the mechanical response of bone. The current study attempts to develop an FE model to describe the bone remodeling process, taking into consideration the activities of osteoclasts and osteoblasts. The mechanical behavior of bone is described by taking into account the bone material fatigue damage accumulation and mineralization. A coupled strain-damage stimulus function is proposed, which controls the level of autocrine and paracrine factors. The cellular behavior is based on Komarova et al.'s (2003) dynamic law, which describes the autocrine and paracrine interactions between osteoblasts and osteoclasts and computes cell population dynamics and changes in bone mass at a discrete site of bone remodeling. Therefore, when an external mechanical stress is applied, bone formation and resorption is governed by cells dynamic rather than adaptive elasticity approaches. The proposed FE model has been implemented in the FE code Abaqus (UMAT routine). An example of human proximal femur is investigated using the model developed. The model was able to predict final human proximal femur adaptation similar to the patterns observed in a human proximal femur. The results obtained reveal complex spatio-temporal bone

  7. Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts.

    PubMed

    Luan, Jun-Bo; Shan, Hong-Wei; Isermann, Philipp; Huang, Jia-Hsin; Lammerding, Jan; Liu, Shu-Sheng; Douglas, Angela E

    2016-06-29

    Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell-cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell-cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends. PMID:27358364

  8. Elastic properties of epithelial cells probed by atomic force microscopy.

    PubMed

    Brückner, Bastian R; Janshoff, Andreas

    2015-11-01

    Cellular mechanics plays a crucial role in many biological processes such as cell migration, cell growth, embryogenesis, and oncogenesis. Epithelia respond to environmental cues comprising biochemical and physical stimuli through defined changes in cell elasticity. For instance, cells can differentiate between certain properties such as viscoelasticity or topography of substrates by adapting their own elasticity and shape. A living cell is a complex viscoelastic body that not only exhibits a shell architecture composed of a membrane attached to a cytoskeleton cortex but also generates contractile forces through its actomyosin network. Here we review cellular mechanics of single cells in the context of epithelial cell layers responding to chemical and physical stimuli. This article is part of a Special Issue entitled: Mechanobiology. PMID:26193077

  9. Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

    PubMed

    Mauger, P-E; Labbé, C; Bobe, J; Cauty, C; Leguen, I; Baffet, G; Le Bail, P-Y

    2009-03-01

    Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

  10. Somatic embryogenesis - Stress-induced remodeling of plant cell fate.

    PubMed

    Fehér, Attila

    2015-04-01

    Plants as sessile organisms have remarkable developmental plasticity ensuring heir continuous adaptation to the environment. An extreme example is somatic embryogenesis, the initiation of autonomous embryo development in somatic cells in response to exogenous and/or endogenous signals. In this review I briefly overview the various pathways that can lead to embryo development in plants in addition to the fertilization of the egg cell and highlight the importance of the interaction of stress- and hormone-regulated pathways during the induction of somatic embryogenesis. Somatic embryogenesis can be initiated in planta or in vitro, directly or indirectly, and the requirement for dedifferentiation as well as the way to achieve developmental totipotency in the various systems is discussed in light of our present knowledge. The initiation of all forms of the stress/hormone-induced in vitro as well as the genetically provoked in planta somatic embryogenesis requires extensive and coordinated genetic reprogramming that has to take place at the chromatin level, as the embryogenic program is under strong epigenetic repression in vegetative plant cells. Our present knowledge on chromatin-based mechanisms potentially involved in the somatic-to-embryogenic developmental transition is summarized emphasizing the potential role of the chromatin to integrate stress, hormonal, and developmental pathways leading to the activation of the embryogenic program. The role of stress-related chromatin reorganization in the genetic instability of in vitro cultures is also discussed. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

  11. Comparison of functional limbal epithelial stem cell isolation methods.

    PubMed

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Dziasko, Marc; Galindo, Sara; Rey, Esther; Herreras, José M; Corrales, Rosa M; Daniels, Julie T; Calonge, Margarita

    2016-05-01

    The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either

  12. Intracellular Ca(2+) remodeling during the phenotypic journey of human coronary smooth muscle cells.

    PubMed

    Muñoz, Eva; Hernández-Morales, Miriam; Sobradillo, Diego; Rocher, Asunción; Núñez, Lucía; Villalobos, Carlos

    2013-11-01

    Vascular smooth muscle cells undergo phenotypic switches after damage which may contribute to proliferative disorders of the vessel wall. This process has been related to remodeling of Ca(2+) channels. We have tested the ability of cultured human coronary artery smooth muscle cells (hCASMCs) to return from a proliferative to a quiescent behavior and the contribution of intracellular Ca(2+) remodeling to the process. We found that cultured, early passage hCASMCs showed a high proliferation rate, sustained increases in cytosolic [Ca(2+)] in response to angiotensin II, residual voltage-operated Ca(2+) entry, increased Stim1 and enhanced store-operated currents. Non-steroidal anti-inflammatory drugs inhibited store-operated Ca(2+) entry and abolished cell proliferation in a mitochondria-dependent manner. After a few passages, hCASMCs turned to a quiescent phenotype characterized by lack of proliferation, oscillatory Ca(2+) response to angiotensin II, increased Ca(2+) store content, enhanced voltage-operated Ca(2+) entry and Cav1.2 expression, and decreases in Stim1, store-operated current and store-operated Ca(2+) entry. We conclude that proliferating hCASMCs return to quiescence and this switch is associated to a remodeling of Ca(2+) channels and their control by subcellular organelles, thus providing a window of opportunity for targeting phenotype-specific Ca(2+) channels involved in proliferation. PMID:24079969

  13. Intracellular Ca(2+) remodeling during the phenotypic journey of human coronary smooth muscle cells.

    PubMed

    Muñoz, Eva; Hernández-Morales, Miriam; Sobradillo, Diego; Rocher, Asunción; Núñez, Lucía; Villalobos, Carlos

    2013-11-01

    Vascular smooth muscle cells undergo phenotypic switches after damage which may contribute to proliferative disorders of the vessel wall. This process has been related to remodeling of Ca(2+) channels. We have tested the ability of cultured human coronary artery smooth muscle cells (hCASMCs) to return from a proliferative to a quiescent behavior and the contribution of intracellular Ca(2+) remodeling to the process. We found that cultured, early passage hCASMCs showed a high proliferation rate, sustained increases in cytosolic [Ca(2+)] in response to angiotensin II, residual voltage-operated Ca(2+) entry, increased Stim1 and enhanced store-operated currents. Non-steroidal anti-inflammatory drugs inhibited store-operated Ca(2+) entry and abolished cell proliferation in a mitochondria-dependent manner. After a few passages, hCASMCs turned to a quiescent phenotype characterized by lack of proliferation, oscillatory Ca(2+) response to angiotensin II, increased Ca(2+) store content, enhanced voltage-operated Ca(2+) entry and Cav1.2 expression, and decreases in Stim1, store-operated current and store-operated Ca(2+) entry. We conclude that proliferating hCASMCs return to quiescence and this switch is associated to a remodeling of Ca(2+) channels and their control by subcellular organelles, thus providing a window of opportunity for targeting phenotype-specific Ca(2+) channels involved in proliferation.

  14. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  15. 5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4(+) T Cells.

    PubMed

    Nestor, Colm E; Lentini, Antonio; Hägg Nilsson, Cathrine; Gawel, Danuta R; Gustafsson, Mika; Mattson, Lina; Wang, Hui; Rundquist, Olof; Meehan, Richard R; Klocke, Bernward; Seifert, Martin; Hauck, Stefanie M; Laumen, Helmut; Zhang, Huan; Benson, Mikael

    2016-07-12

    5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4(+) memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) T cell biology in humans, with important implications for gene regulation and lineage commitment.

  16. 5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4(+) T Cells.

    PubMed

    Nestor, Colm E; Lentini, Antonio; Hägg Nilsson, Cathrine; Gawel, Danuta R; Gustafsson, Mika; Mattson, Lina; Wang, Hui; Rundquist, Olof; Meehan, Richard R; Klocke, Bernward; Seifert, Martin; Hauck, Stefanie M; Laumen, Helmut; Zhang, Huan; Benson, Mikael

    2016-07-12

    5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4(+) memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) T cell biology in humans, with important implications for gene regulation and lineage commitment. PMID:27346350

  17. Cell elongation is key to in silico replication of in vitro vasculogenesis and subsequent remodeling

    PubMed Central

    Merks, Roeland M.H.; Brodsky, Sergey V.; Goligorksy, Michael S.; Newman, Stuart A.; Glazier, James A.

    2008-01-01

    Vasculogenesis, the de novo growth of the primary vascular network from initially dispersed endothelial cells, is the first step in the development of the circulatory system in vertebrates. In the first stages of vasculogenesis, endothelial cells elongate and form a network-like structure, called the primary capillary plexus, which subsequently remodels, with the size of the vacancies between ribbons of endothelial cells coarsening over time. To isolate such intrinsic morphogenetic ability of endothelial cells from its regulation by long-range guidance cues and additional cell types, we use an in vitro model of human umbilical vein endothelial cells (HUVEC) in Matrigel. This quasi-two-dimensional endothelial cell culture model would most closely correspond to vasculogenesis in flat areas of the embryo like the yolk sac. Several studies have used continuum mathematical models to explore in vitro vasculogenesis: such models describe cell ensembles but ignore the endothelial cells’ shapes and active surface fluctuations. While these models initially reproduce vascular-like morphologies, they eventually stabilize into a disconnected pattern of vascular “islands.” Also, they fail to reproduce temporally correct network coarsening. Using a cell-centered computational model, we show that the endothelial cells’ elongated shape is key to correct spatiotemporal in silico replication of stable vascular network growth. We validate our simulation results against HUVEC cultures using time-resolved image analysis and find that our simulations quantitatively reproduce in vitro vasculogenesis and subsequent in vitro remodeling. PMID:16325173

  18. Structure of Pneumococcal Peptidoglycan Hydrolase LytB Reveals Insights into the Bacterial Cell Wall Remodeling and Pathogenesis*

    PubMed Central

    Bai, Xiao-Hui; Chen, Hui-Jie; Jiang, Yong-Liang; Wen, Zhensong; Huang, Yubin; Cheng, Wang; Li, Qiong; Qi, Lei; Zhang, Jing-Ren; Chen, Yuxing; Zhou, Cong-Zhao

    2014-01-01

    Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases. PMID:25002590

  19. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

  20. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation. PMID:27520504

  1. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

    PubMed Central

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs. PMID:27409796

  2. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells.

    PubMed

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  3. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells

    PubMed Central

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  4. Computational Modeling Predicts Simultaneous Targeting of Fibroblasts and Epithelial Cells Is Necessary for Treatment of Pulmonary Fibrosis

    PubMed Central

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; Linderman, Jennifer J.; Moore, Bethany B.; Kirschner, Denise E.

    2016-01-01

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGF-β1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. Thus, a two-hit therapeutic intervention strategy

  5. Computational Modeling Predicts Simultaneous Targeting of Fibroblasts and Epithelial Cells Is Necessary for Treatment of Pulmonary Fibrosis.

    PubMed

    Warsinske, Hayley C; Wheaton, Amanda K; Kim, Kevin K; Linderman, Jennifer J; Moore, Bethany B; Kirschner, Denise E

    2016-01-01

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGF-β1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. Thus, a two-hit therapeutic intervention strategy

  6. Computational modeling predicts simultaneous targeting of fibroblasts and epithelial cells is necessary for treatment of pulmonary fibrosis

    DOE PAGESBeta

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; Linderman, Jennifer J.; Moore, Bethany B.; Kirschner, Denise E.

    2016-06-23

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited withmore » enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGFβ1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. In conclusion, a two-hit therapeutic

  7. Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts

    PubMed Central

    Luan, Jun-Bo; Shan, Hong-Wei; Isermann, Philipp; Huang, Jia-Hsin; Lammerding, Jan; Liu, Shu-Sheng; Douglas, Angela E.

    2016-01-01

    Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell–cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell–cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends. PMID:27358364

  8. Mitochondrial dysfunction remodels one-carbon metabolism in human cells.

    PubMed

    Bao, Xiaoyan Robert; Ong, Shao-En; Goldberger, Olga; Peng, Jun; Sharma, Rohit; Thompson, Dawn A; Vafai, Scott B; Cox, Andrew G; Marutani, Eizo; Ichinose, Fumito; Goessling, Wolfram; Regev, Aviv; Carr, Steven A; Clish, Clary B; Mootha, Vamsi K

    2016-01-01

    Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis. PMID:27307216

  9. Mitochondrial dysfunction remodels one-carbon metabolism in human cells

    PubMed Central

    Bao, Xiaoyan Robert; Ong, Shao-En; Goldberger, Olga; Peng, Jun; Sharma, Rohit; Thompson, Dawn A; Vafai, Scott B; Cox, Andrew G; Marutani, Eizo; Ichinose, Fumito; Goessling, Wolfram; Regev, Aviv; Carr, Steven A; Clish, Clary B; Mootha, Vamsi K

    2016-01-01

    Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.10575.001 PMID:27307216

  10. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  11. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  12. Studies in human skin epithelial cell carcinogenesis

    SciTech Connect

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo(a)pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and /sup 32/P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the /sup 32/P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts.

  13. Hertwig's epithelial root sheath cells do not transform into cementoblasts in rat molar cementogenesis.

    PubMed

    Yamamoto, Tsuneyuki; Takahashi, Shigeru

    2009-12-01

    It is generally accepted that cementoblasts originate in the process of differentiation of the mesenchymal cells of the dental follicle. Recently, a different hypothesis for the origin of cementoblasts has been proposed. Hertwig's epithelial root sheath cells undergo the epithelial-mesenchymal transformation to differentiate into cementoblasts. To elucidate whether the epithelial-mesenchymal transformation occurs in the epithelial sheath, developing rat molars were examined by keratin-vimentin and Runx2 (runt-related transcription factor 2)-keratin double immunostaining. In both acellular and cellular cementogenesis, epithelial sheath and epithelial cells derived from the epithelial sheath expressed keratin, but did not express vimentin or Runx2. Dental follicle cells and cementoblasts, however, expressed vimentin and Runx2, but did not express keratin. No cells showed coexisting keratin-vimentin or Runx2-keratin staining. These findings suggest that there is no intermediate phenotype transforming epithelial to mesenchymal cells, and that epithelial sheath cells do not generate mineralized tissue. This study concludes that the epithelial-mesenchymal transformation does not occur in Hertwig's epithelial root sheath in rat acellular or cellular cementogenesis and that the dental follicle is the origin of cementoblasts, as has been proposed in the original hypothesis. PMID:19716687

  14. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  15. Thymic epithelial cells: working class heroes for T cell development and repertoire selection.

    PubMed

    Anderson, Graham; Takahama, Yousuke

    2012-06-01

    The thymus represents an epithelial-mesenchymal tissue, anatomically structured into discrete cortical and medullary regions that contain phenotypically and functionally distinct stromal cells, as well as thymocytes at defined stages of maturation. The stepwise progression of thymocyte development seems to require serial migration through these distinct thymic regions, where interactions with cortical thymic epithelial cell (cTEC) and medullary thymic epithelial cell (mTEC) subsets take place. Recent work on TEC subsets provides insight into T cell development and selection, such as the importance of tumour necrosis factor (TNF) receptor superfamily members in thymus medulla development, and the specialised antigen processing/presentation capacity of the thymic cortex for positive selection. Here, we summarise current knowledge on the development and function of the thymic microenvironment, paying particular attention to the cortical and medullary epithelial compartments.

  16. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  17. REMOD: A Tool for Analyzing and Remodeling the Dendritic Architecture of Neural Cells

    PubMed Central

    Bozelos, Panagiotis; Stefanou, Stefanos S.; Bouloukakis, Georgios; Melachrinos, Constantinos; Poirazi, Panayiota

    2016-01-01

    Dendritic morphology is a key determinant of how individual neurons acquire a unique signal processing profile. The highly branched dendritic structure that originates from the cell body, explores the surrounding 3D space in a fractal-like manner, until it reaches a certain amount of complexity. Its shape undergoes significant alterations under various physiological or neuropathological conditions. Yet, despite the profound effect that these alterations can have on neuronal function, the causal relationship between the two remains largely elusive. The lack of a systematic approach for remodeling neural cells and their dendritic trees is a key limitation that contributes to this problem. Such causal relationships can be inferred via the use of large-scale neuronal models whereby the anatomical plasticity of neurons is accounted for, in order to enhance their biological relevance and hence their predictive performance. To facilitate this effort, we developed a computational tool named REMOD that allows the structural remodeling of any type of virtual neuron. REMOD is written in Python and can be accessed through a dedicated web interface that guides the user through various options to manipulate selected neuronal morphologies. REMOD can also be used to extract meaningful morphology statistics for one or multiple reconstructions, including features such as sholl analysis, total dendritic length and area, path length to the soma, centrifugal branch order, diameter tapering and more. As such, the tool can be used both for the analysis and/or the remodeling of neuronal morphologies of any type. PMID:26778971

  18. Luteolin Ameliorates Hypertensive Vascular Remodeling through Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells

    PubMed Central

    Su, Jie; Xu, Han-Ting; Yu, Jing-Jing; Gao, Jian-Li; Lei, Jing; Yin, Qiao-Shan; Li, Bo; Pang, Min-Xia; Su, Min-Xia; Mi, Wen-Jia; Chen, Su-Hong; Lv, Gui-Yuan

    2015-01-01

    Objectives. Preliminary researches showed that luteolin was used to treat hypertension. However, it is still unclear whether luteolin has effect on the hypertensive complication such as vascular remodeling. The present study was designed to investigate the effect of luteolin on the hypertensive vascular remodeling and its molecular mechanism. Method and Results. We evaluated the effect of luteolin on aorta thickening of hypertension in spontaneous hypertensive rats (SHRs) and found that luteolin could significantly decrease the blood pressure and media thickness of aorta in vivo. Luteolin could inhibit angiotensin II- (Ang II-) induced proliferation and migration of vascular smooth muscle cells (VSMCs). Dichlorofluorescein diacetate (DCFH-DA) staining result showed that luteolin reduced Ang II-stimulated ROS production in VSMCs. Furthermore, western blot and gelatin zymography results showed that luteolin treatment leaded to a decrease in ERK1/2, p-ERK1/2, p-p38, MMP2, and proliferating cell nuclear antigen (PCNA) protein level. Conclusion. These data support that luteolin can ameliorate hypertensive vascular remodeling by inhibiting the proliferation and migration of Ang II-induced VSMCs. Its mechanism is mediated by the regulation of MAPK signaling pathway and the production of ROS. PMID:26495010

  19. REMOD: A Tool for Analyzing and Remodeling the Dendritic Architecture of Neural Cells.

    PubMed

    Bozelos, Panagiotis; Stefanou, Stefanos S; Bouloukakis, Georgios; Melachrinos, Constantinos; Poirazi, Panayiota

    2015-01-01

    Dendritic morphology is a key determinant of how individual neurons acquire a unique signal processing profile. The highly branched dendritic structure that originates from the cell body, explores the surrounding 3D space in a fractal-like manner, until it reaches a certain amount of complexity. Its shape undergoes significant alterations under various physiological or neuropathological conditions. Yet, despite the profound effect that these alterations can have on neuronal function, the causal relationship between the two remains largely elusive. The lack of a systematic approach for remodeling neural cells and their dendritic trees is a key limitation that contributes to this problem. Such causal relationships can be inferred via the use of large-scale neuronal models whereby the anatomical plasticity of neurons is accounted for, in order to enhance their biological relevance and hence their predictive performance. To facilitate this effort, we developed a computational tool named REMOD that allows the structural remodeling of any type of virtual neuron. REMOD is written in Python and can be accessed through a dedicated web interface that guides the user through various options to manipulate selected neuronal morphologies. REMOD can also be used to extract meaningful morphology statistics for one or multiple reconstructions, including features such as sholl analysis, total dendritic length and area, path length to the soma, centrifugal branch order, diameter tapering and more. As such, the tool can be used both for the analysis and/or the remodeling of neuronal morphologies of any type. PMID:26778971

  20. Cell-Envelope Remodeling as a Determinant of Phenotypic Antibacterial Tolerance in Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    The mechanisms that lead to phenotypic antibacterial tolerance in bacteria remain poorly understood. We investigate whether changes in NaCl concentration toward physiologically higher values affect antibacterial efficacy against Mycobacterium tuberculosis (Mtb), the causal agent of human tuberculosis. Indeed, multiclass phenotypic antibacterial tolerance is observed during Mtb growth in physiologic saline. This includes changes in sensitivity to ethionamide, ethambutol, d-cycloserine, several aminoglycosides, and quinolones. By employing organism-wide metabolomic and lipidomic approaches combined with phenotypic tests, we identified a time-dependent biphasic adaptive response after exposure of Mtb to physiological levels of NaCl. A first rapid, extensive, and reversible phase was associated with changes in core and amino acid metabolism. In a second phase, Mtb responded with a substantial remodelling of plasma membrane and outer lipid membrane composition. We demonstrate that phenotypic tolerance at physiological concentrations of NaCl is the result of changes in plasma and outer membrane lipid remodeling and not changes in core metabolism. Altogether, these results indicate that physiologic saline-induced antibacterial tolerance is kinetically coupled to cell envelope changes and demonstrate that metabolic changes and growth arrest are not the cause of phenotypic tolerance observed in Mtb exposed to physiologic concentrations of NaCl. Importantly, this work uncovers a role for bacterial cell envelope remodeling in antibacterial tolerance, alongside well-documented allterations in respiration, metabolism, and growth rate. PMID:27231718

  1. Incremental responses to light recorded from pigment epithelial cells and horizontal cells of the cat retina

    PubMed Central

    Steinberg, Roy H.

    1971-01-01

    1. Rod-dependent incremental responses were recorded intracellularly in both pigment epithelial cells and horizontal cells of the cat retina. They were elicited by test flashes which were superimposed on background flashes after a delay. 2. In pigment epithelial cells smaller test responses were produced as background intensity was raised. The incremental sensitivity function was linear for about 1·4 log units, with a slope of 0·86, and the approach of saturation occurred at about 2·5 log td scotopic. 3. The amplitude of pigment epithelial test responses could be estimated from the dark-adapted amplitude—log intensity function obtained with single flashes. Test flashes produced the voltage increment predicted by the slope of this function just above the point on the curve equal to the background intensity. The pigment epithelial response to a test flash, therefore, is the response expected if the background were presented alone and made more intense by the amount of the test flash. 4. Rod-dependent incremental sensitivity functions of horizontal cells closely resembled the ones obtained from pigment epithelial cells. 5. It was concluded that the adaptive effects observed in pigment epithelial cells originated in individual rods. These effects arose from the compressive nature of the dark-adapted amplitude—intensity function. In horizontal cell responses these effects may be modified by the failure of the background response to maintain its initial voltage. PMID:5571955

  2. Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells

    PubMed Central

    Hishikawa, Daisuke; Hashidate, Tomomi; Shimizu, Takao; Shindou, Hideo

    2014-01-01

    Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained. PMID:24646950

  3. Differentiation potentials of perivascular cells in the bone tissue remodeling zones under microgravity

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia; Katkova, Olena

    Adaptive remodeling processes in the skeleton bones occur in the close topographical interconnection with blood capillaries followed by perivascular cells. Radioautographic studies with 3Н- thymidine (Kimmel D.B., Fee W.S., 1980; Rodionova N.V., 1989, 2006) has shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic ones. Using electron microscopy and cytochemistry we studied perivsacular cells in metaphysis of the rats femoral bones under conditions of modeling microgravity (28 days duration) and in femoral bonеs metaphyses of rats flown on board of the space laboratory (Spacelab - 2) It was revealed that population of the perivascular cells is not homogeneous in adaptive zones of the remodeling in both control and test groups (lowering support loading). This population comprises adjacent to endothelium little differentiated forms and isolated cells with differentiation features (specific volume of rough endoplasmic reticulum in cytoplasm is increased). Majority of the perivascular cells in the control group reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In little differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of animals under microgravitaty reaction to the alkaline phosphatase is registered not for all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. There is also visible trend of individual alkaline phosphatase containing perivascular cells amounts decrease (i.e. osteogenic cells-precursors). Under microgravity some little differentiated perivascular cells reveal destruction signs. Found decrease trend of the alkaline phosphatase containing cells (i.e. osteogenic cells) number in

  4. Oral microbial biofilm stimulation of epithelial cell responses.

    PubMed

    Peyyala, Rebecca; Kirakodu, Sreenatha S; Novak, Karen F; Ebersole, Jeffrey L

    2012-04-01

    Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria.

  5. Human epithelial cell cultures from superficial limbal explants

    PubMed Central

    Basli, E.; Goldschmidt, P.; Pecha, F.; Chaumeil, C.; Laroche, L.; Borderie, V.

    2011-01-01

    Purpose To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer. Methods Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR). Results The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm2 and 429 µm2, and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human

  6. Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

    PubMed

    Martínez-Martínez, Eduardo; Uribe-Querol, Eileen; Galván-Hernández, Claudio I; Gutiérrez-Ospina, Gabriel

    2016-01-01

    We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology. PMID:27431250

  7. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  8. 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.

  9. DipM, a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus.

    PubMed

    Möll, Andrea; Schlimpert, Susan; Briegel, Ariane; Jensen, Grant J; Thanbichler, Martin

    2010-07-01

    In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C-terminal lysostaphin-like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.

  10. Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

    PubMed Central

    Cobo, Teresa; Viloria, Cristina G.; Solares, Laura; Fontanil, Tania; González-Chamorro, Elena; De Carlos, Félix; Cobo, Juan; Cal, Santiago; Obaya, Alvaro J.

    2016-01-01

    Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology. PMID:26809067

  11. Changes in the population of perivascular cells in the bone tissue remodeling zones under microgravity

    NASA Astrophysics Data System (ADS)

    Katkova, Olena; Rodionova, Natalia; Shevel, Ivan

    2016-07-01

    Microgravity and long-term hypokinesia induce reduction both in bone mass and mineral saturation, which can lead to the development of osteoporosis and osteopenia. (Oganov, 2003). Reorganizations and adaptive remodeling processes in the skeleton bones occur in the topographical interconnection with blood capillaries and perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel, Fee, 1980; Rodionova, 1989, 2006) have shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic. Hence the study of populations of perivascular stromal cells in areas of destructive changes is actual. Perivascular cells from metaphysis of the rat femoral bones under conditions of modeling microgravity were studied using electron microscopy and cytochemistry (hindlimb unloading, 28 days duration) and biosatellite «Bion-M1» (duration of flight from April 19 till May 19, 2013 on C57, black mice). It was revealed that both control and test groups populations of the perivascular cells are not homogeneous in remodeling adaptive zones. These populations comprise of adjacent to endothelium poorly differentiated forms and isolated cells with signs of differentiation (specific increased volume of rough endoplasmic reticulum in cytoplasm). Majority of the perivascular cells in the control group (modeling microgravity) reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In poorly differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of experimental animals reaction to the alkaline phosphatase is registered not in all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. Under microgravity some poorly differentiated perivascular

  12. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    NASA Astrophysics Data System (ADS)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-02-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  13. Estrogen Inhibits Mast Cell Chymase Release to Prevent Pressure Overload-Induced Adverse Cardiac Remodeling

    PubMed Central

    Li, Jianping; Jubair, Shaiban; Janicki, Joseph S.

    2014-01-01

    Estrogen regulation of myocardial chymase and chymase effects on cardiac remodeling are unknown. To test the hypothesis that estrogen prevents pressure overload-induced adverse cardiac remodeling by inhibiting mast cell chymase release, transverse aortic constriction or sham surgery was performed in 7-week-old intact and ovariectomized rats. Three days prior to creating the constriction, additional groups of ovariectomized rats began receiving 17β-Estradiol, a chymase inhibitor, or a mast cell stabilizer. Left ventricular function, cardiomyocyte size, collagen volume fraction, mast cell density and degranulation, and myocardial and plasma chymase levels were assessed 18 days post-surgery. Aortic constriction resulted in ventricular hypertrophy in intact and ovariectomized groups while collagen volume fraction was increased only in ovariectomized rats. Chymase protein content was increased by aortic constriction in the intact and ovariectomized groups with the magnitude of the increase being greater in ovariectomized rats. Mast cell density and degranulation, plasma chymase levels and myocardial active transforming growth factor- 1 levels were increased by aortic constriction only in ovariectomized rats. Estrogen replacement markedly attenuated the constriction-increased myocardial chymase, mast cell density and degranulation, plasma chymase and myocardial active transforming growth factor- 1 as well as prevented ventricular hypertrophy and increased collagen volume fraction. Chymostatin attenuated the aortic constriction induced ventricular hypertrophy and collagen volume fraction in the ovariectomized rats similar to that achieved by estrogen replacement. Nedocromil yielded similar effects except for the reduction of chymase content. We conclude that the estrogen-inhibited release of mast cell chymase is responsible for the cardioprotection against transverse aortic constriction-induced adverse cardiac remodeling. PMID:25403608

  14. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  15. Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

    PubMed

    Marelli, Benedetto; Le Nihouannen, Damien; Hacking, S Adam; Tran, Simon; Li, Jingjing; Murshed, Monzur; Doillon, Charles J; Ghezzi, Chiara E; Zhang, Yu Ling; Nazhat, Showan N; Barralet, Jake E

    2015-06-01

    Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery. PMID:25907046

  16. Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

    PubMed

    Marelli, Benedetto; Le Nihouannen, Damien; Hacking, S Adam; Tran, Simon; Li, Jingjing; Murshed, Monzur; Doillon, Charles J; Ghezzi, Chiara E; Zhang, Yu Ling; Nazhat, Showan N; Barralet, Jake E

    2015-06-01

    Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery.

  17. Interleukin-6 induces epithelial-mesenchymal transition in human intrahepatic biliary epithelial cells

    PubMed Central

    JIANG, GUI-XING; CAO, LI-PING; KANG, PENG-CHENG; ZHONG, XIANG-YU; LIN, TIAN-YU; CUI, YUN-FU

    2016-01-01

    The aim of the present study was to determine the role of interleukin-6 (IL-6) in the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cell (HIBEC) lines in vitro. HIBECs were stimulated with IL-6 at concentrations of 0, 10, 20, 50 and 100 µg/l for 24 h. A wound healing and Transwell assay were performed to determine the migratory and invasive capacity of HIBECs, respectively. Following 24 h of incubation, IL-6 at 10 and 20 µg/l significantly increased the number of migrated and invaded cells (P<0.05), while stimulation with 50 and 100 µg/l IL-6 resulted in a further increase of the migratory and invasive capacity compared to that in all other groups (P<0.05). Furthermore, reverse-transcription quantitative polymerase chain reaction and western blot analyses were used to detect the mRNA and protein expression of EMT markers E-cadherin and vimentin in HIBECs. Decreased mRNA levels of E-cadherin accompanied by higher mRNA levels of vimentin were observed in the 10, 20, 50, 100 µg/l IL-6 groups compared to those in the 0 µg/l group (all P<0.05). Furthermore, the protein expression of E-cadherin was decreased, while that of vimentin was increased in the 50 and 100 µg/l IL-6 groups compared to those in the 0, 10 and 20 µg/l IL-6 groups (all P<0.05). The present study therefore indicated that IL-6 promoted the process of EMT in HIBECs as characterized by increased migration and invasion of HIBECs and the typical changes in mRNA and protein expression of the EMT markers E-cadherin and vimentin. PMID:26708270

  18. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    PubMed

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  19. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.

  20. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  1. Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

    NASA Astrophysics Data System (ADS)

    Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

    2006-07-01

    A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

  2. Dynamic Reorganization and Enzymatic Remodeling of Type IV Collagen at Cell-Biomaterial Interface.

    PubMed

    Coelho, N M; Llopis-Hernández, V; Salmerón-Sánchez, M; Altankov, G

    2016-01-01

    Vascular basement membrane remodeling involves assembly and degradation of its main constituents, type IV collagen (Col IV) and laminin, which is critical during development, angiogenesis, and tissue repair. Remodeling can also occur at cell-biomaterials interface altering significantly the biocompatibility of implants. Here we describe the fate of adsorbed Col IV in contact with endothelial cells adhering on positively charged NH2 or hydrophobic CH3 substrata, both based on self-assembly monolayers (SAMs) and studied alone or mixed in different proportions. AFM studies revealed distinct pattern of adsorbed Col IV, varying from single molecular deposition on pure NH2 to network-like assembly on mixed SAMs, turning to big globular aggregates on bare CH3. Human umbilical endothelial cells (HUVECs) interact better with Col IV adsorbed as single molecules on NH2 surface and readily rearrange it in fibril-like pattern that coincide with secreted fibronectin fibrils. The cells show flattened morphology and well-developed focal adhesion complexes that are rich on phosphorylated FAK while expressing markedly low pericellular proteolytic activity. Conversely, on hydrophobic CH3 substrata HUVECs showed abrogated spreading and FAK phosphorylation, combined with less reorganization of the aggregated Col IV and significantly increased proteolytic activity. The later involves both MMP-2 and MMP-9, as measured by zymography and FITC-Col IV release. The mixed SAMs support intermediate remodeling activity. Taken together these results show that chemical functionalization combined with Col IV preadsorption provides a tool for guiding the endothelial cells behavior and pericellular proteolytic activity, events that strongly affect the fate of cardiovascular implants. PMID:27567485

  3. Sensing, signaling and sorting events in kidney epithelial cell physiology.

    PubMed

    Brown, Dennis; Breton, Sylvie; Ausiello, Dennis A; Marshansky, Vladimir

    2009-03-01

    The kidney regulates body fluid, ion and acid/base homeostasis through the interaction of a host of channels, transporters and pumps within specific tubule segments, specific cell types and specific plasma membrane domains. Furthermore, renal epithelial cells have adapted to function in an often unique and challenging environment that includes high medullary osmolality, acidic pHs, variable blood flow and constantly changing apical and basolateral 'bathing' solutions. In this review, we focus on selected protein trafficking events by which kidney epithelial cells regulate body fluid, ion and acid-base homeostasis in response to changes in physiological conditions. We discuss aquaporin 2 and G-protein-coupled receptors in fluid and ion balance, the vacuolar H(+)-adenosine triphosphatase (V-ATPase) and intercalated cells in acid/base regulation and acidification events in the proximal tubule degradation pathway. Finally, in view of its direct role in vesicle trafficking that we outline in this study, we propose that the V-ATPase itself should, under some circumstances, be considered a fourth category of vesicle 'coat' protein (COP), alongside clathrin, caveolin and COPs. PMID:19170982

  4. Sensing, signaling and sorting events in kidney epithelial cell physiology

    PubMed Central

    Brown, Dennis; Breton, Sylvie; Ausiello, Dennis A.; Marshansky, Vladimir

    2010-01-01

    The kidney regulates body fluid, ion, and acid/base homeostasis via the interaction of a host of channels, transporters, and pumps within specific tubule segments, specific cell types, and specific plasma membrane domains. Furthermore, renal epithelial cells have adapted to function in an often unique and challenging environment that includes high medullary osmolality, acidic pHs, variable blood flow and constantly changing apical and basolateral “bathing” solutions. In this review, we focus on selected protein trafficking events by which kidney epithelial cells regulate body fluid, ion and acid-base homeostasis in response to changes in physiological conditions. We discuss aquaporin 2 and G-protein coupled receptors (GPCRs) in fluid and ion balance, the V-ATPase and intercalated cells (IC) in acid/base regulation, and acidification events in the proximal tubule degradation pathway. Finally, in view of its direct role in vesicle trafficking that we outline here, we propose that the V-ATPase itself should, under some circumstances, be considered as a fourth category of vesicle “coat” protein, alongside clathrin, caveolin, and COPs. PMID:19170982

  5. Organization of the cytokeratin network in an epithelial cell.

    PubMed

    Portet, Stéphanie; Arino, Ovide; Vassy, Jany; Schoëvaërt, Damien

    2003-08-01

    The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.

  6. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

    PubMed

    Lamberti, Yanina; Gorgojo, Juan; Massillo, Cintia; Rodriguez, Maria E

    2013-12-01

    Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

  7. MicroRNA-146a modulates human bronchial epithelial cell survival in response to the cytokine-induced apoptosis

    SciTech Connect

    Liu Xiangde Nelson, Amy; Wang Xingqi; Kanaji, Nobuhiro; Kim, Miok; Sato, Tadashi; Nakanishi, Masanori; Li Yingji; Sun Jianhong; Michalski, Joel; Patil, Amol; Basma, Hesham; Rennard, Stephen I.

    2009-02-27

    MicroRNA plays an important role in cell differentiation, proliferation and cell death. The current study found that miRNA-146a was up-regulated in human bronchial epithelial cells (HBECs) in response to stimulation by TGF-ss1 plus cytomix (a mixture of IL-1ss, IFN-{gamma} and TNF-{alpha}). TGF-ss1 plus cytomix (TCM) induced apoptosis in HBECs (3.4 {+-} 0.6% of control vs 83.1 {+-} 4.0% of TCM treated cells, p < 0.01), and this was significantly blocked by the miRNA-146a mimic (8.8 {+-} 1.5%, p < 0.01). In contrast, a miRNA-146a inhibitor had only a modest effect on cell survival but appeared to augment the induction of epithelial-mesenchymal transition (EMT) in response to the cytokines. The MicroRNA-146a mimic appears to modulate HBEC survival through a mechanism of up-regulating Bcl-XL and STAT3 phosphorylation, and by this mechanism it could contribute to tissue repair and remodeling.

  8. Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles

    PubMed Central

    Tong, Peter; Khayat, Zayna A.; Huang, Carol; Patel, Nish; Ueyama, Atsunori; Klip, Amira

    2001-01-01

    Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular compartment to the cell surface; this phenomenon is defective in type 2 diabetes. Here we examine the involvement of actin filaments in GLUT4 translocation and their possible defects in insulin resistance, using L6 myotubes expressing myc-tagged GLUT4. Insulin caused membrane ruffling, a dynamic distortion of the myotube dorsal surface. Fluorescence microscopy and immunogold staining of surface GLUT4myc coupled to backscatter electron microscopy revealed a high density of this protein in membrane ruffles. The t-SNAREs syntaxin4 and SNAP-23 were also abundant in these regions. Below the membrane, GLUT4 and the vesicular protein VAMP2, but not VAMP3, colocalized with the actin structures supporting the membrane ruffles. GLUT4myc externalization and membrane ruffles were reduced by jasplakinolide and by swinholide-A, drugs that affect actin filament stability and prevent actin branching, respectively. Insulin resistance generated by prolonged (24 hours) exposure of myotubes to high glucose and insulin diminished the acute insulin-dependent remodeling of cortical actin and GLUT4myc translocation, reminiscent of the effect of swinholide-A. We propose that GLUT4 vesicle incorporation into the plasma membrane involves insulin-dependent cortical actin remodeling and that defective actin remodeling contributes to insulin resistance. PMID:11489930

  9. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    PubMed

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  10. Relation of smoking to immunoreactive endothelin in the bronchiolar epithelial cells.

    PubMed Central

    Shokeir, M. O.; Paré, P.; Wright, J. L.

    1994-01-01

    BACKGROUND--Endothelin is a potent bronchoconstrictor which appears to be important in asthma. To ascertain whether cigarette smoking is associated with any alteration in the proportion of bronchiolar epithelial cells which express endothelin immunoreactivity, the airways in the lungs of non-smokers and smokers were analysed. Since an increase in immunoreactivity has been found in the bronchial epithelial cells of asthmatic subjects, cigarette smokers with and without evidence of airway hyperresponsiveness were also selected. METHODS--A point counting method which examined the proportion of endothelin immunoreactive epithelial cells in membranous and respiratory bronchioles was used. RESULTS--Neither smoking itself nor evidence of airway hyperresponsiveness altered the percentage of endothelin immunoreactive epithelial cells in the membraneous and respiratory bronchioles. CONCLUSIONS--Cigarette smoke does not induce endothelin production in bronchiolar epithelial cells, and the airway hyperresponsiveness seen in some patients with lung disease induced by cigarette smoking is not related to exaggerated endothelin production in epithelial cells. PMID:8091324

  11. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  12. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  13. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  14. Plasticity of epithelial stem cells in tissue regeneration

    PubMed Central

    Blanpain, Cédric; Fuchs, Elaine

    2015-01-01

    Tissues rely upon stem cells for homeostasis and repair. Recent studies show that the fate and multilineage potential of epithelial stem cells can change depending on whether a stem cell exists within its resident niche and responds to normal tissue homeostasis, whether it is mobilized to repair a wound, or whether it is taken from its niche and challenged to de novo tissue morphogenesis after transplantation. In this Review, we discuss how different populations of naturally lineage-restricted stem cells and committed progenitors can display remarkable plasticity and reversibility and reacquire long-term self-renewing capacities and multilineage differentiation potential during physiological and regenerative conditions. We also discuss the implications of cellular plasticity for regenerative medicine and for cancer. PMID:24926024

  15. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

    PubMed Central

    Choi, Kwang-Hwan; Park, Jin-Kyu; Son, Dongchan; Hwang, Jae Yeon; Lee, Dong-Kyung; Ka, Hakhyun; Park, Joonghoon; Lee, Chang-Kyu

    2016-01-01

    Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig. PMID:27336671

  16. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig.

    PubMed

    Choi, Kwang-Hwan; Park, Jin-Kyu; Son, Dongchan; Hwang, Jae Yeon; Lee, Dong-Kyung; Ka, Hakhyun; Park, Joonghoon; Lee, Chang-Kyu

    2016-01-01

    Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig. PMID:27336671

  17. Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

    PubMed Central

    1994-01-01

    Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed. PMID:7798314

  18. 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.

  19. The Emerging Prominence of the Cardiac Mast Cell as a Potent Mediator of Adverse Myocardial Remodeling

    PubMed Central

    Janicki, Joseph S.; Brower, Gregory L.; Levick, Scott P.

    2015-01-01

    Cardiac mast cells store and release a variety of biologically active mediators, several of which have been implicated in the activation of matrix metalloproteinases in the volume-overloaded heart, while others are involved in the fibrotic process in pressure-overloaded hearts. Increased numbers of mast cells have been reported in explanted human hearts with dilated cardiomyopathy and in animal models of experimentally induced hypertension, myocardial infarction, and chronic cardiac volume overload. Also, there is evolving evidence implicating the cardiac mast cell as having a major role in the adverse remodeling underlying these cardiovascular disorders. Thus, the cardiac mast cell is the focus of this chapter that begins with a historical background, followed by sections on methods for their isolation and characterization, endogenous secretagogues, phenotype, and ability of estrogen to alter their phenotype so as to provide cardioprotection. Finally the role of mast cells in myocardial remodeling secondary to a sustained cardiac volume overload, hypertension, and ischemic injury and future research directions are discussed. PMID:25388248

  20. Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    SciTech Connect

    Zitta, Karina; Brandt, Berenice; Wuensch, Annegret; Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens; Albrecht, Martin

    2010-09-03

    Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferation of pig heart cells in-vitro. {yields} IL-1{beta} increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. {yields} IL-1{beta} may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1{beta} is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1{beta} on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1{beta}. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1{beta} resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1{beta} plays a major role in the events of tissue remodelling in the heart. Combined

  1. In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

    PubMed Central

    Hu, Han; Wang, Jiaqi; Bu, Dengpan; Wei, Hongyang; Zhou, Linyun; Li, Fadi; Loor, Juan J.

    2009-01-01

    Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs). PMID:19888476

  2. Cell crawling mediates collective cell migration to close undamaged epithelial gaps

    PubMed Central

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C.; Sheetz, Michael P.; Trepat, Xavier; Ladoux, Benoît

    2012-01-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  3. Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

    PubMed

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C; Sheetz, Michael P; Trepat, Xavier; Ladoux, Benoît

    2012-07-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  4. Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

    PubMed Central

    Pantenburg, Birte; Castellanos-Gonzalez, Alejandro; Dann, Sara M.; Connelly, Rhykka L.; Lewis, Dorothy E.; Ward, Honorine D.; Clinton White, A.

    2010-01-01

    Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4+ T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8+ T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8+ T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8+ T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite. PMID:20348507

  5. Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling.

    PubMed

    Warncke, Jan D; Vakonakis, Ioannis; Beck, Hans-Peter

    2016-12-01

    During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family. PMID:27582258

  6. 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. PMID:27263094

  7. Contacts between pigmented retina epithelial cells in culture.

    PubMed

    Middleton, C A; Pegrum, S M

    1976-11-01

    The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.

  8. Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

    PubMed

    Chen, Xiuping; Guo, Feiye; LeBlanc, Michelle E; Ding, Ying; Zhang, Chenming; Shakya, Akhalesh; Li, Wei

    2016-08-01

    Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.

  9. Barrier Epithelial Cells and the Control of Type 2 Immunity.

    PubMed

    Hammad, Hamida; Lambrecht, Bart N

    2015-07-21

    Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease.

  10. Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

    PubMed

    Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

    2016-05-01

    Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

  11. Expression of tracheal antimicrobial peptide in bovine mammary epithelial cells.

    PubMed

    López-Meza, Joel E; Gutiérrez-Barroso, Angelina; Ochoa-Zarzosa, Alejandra

    2009-08-01

    The production of antimicrobial peptides is an important key of innate immunity. Tracheal antimicrobial peptide (TAP) expression has been reported in bovine tracheal epithelial cells and it can be modulated by bacterial infection or bacterial components. In mammary gland TAP expression has been reported, but the cell type that produces it is unknown. The objective of this work was to evaluate if bovine mammary epithelial cells (bMEC) express TAP mRNA, and evaluate the regulation of its expression in response to Staphylococcus aureus infection, bovine prolactin (bPRL) or acetyl salicylic acid (ASA). By retrotranscription and PCR, we demonstrated that bMEC express TAP mRNA. bMEC infected with live S. aureus down-regulates TAP expression, whereas the challenge with gentamicin-killed S. aureus up-regulates it. Also, bPRL do not significantly modify TAP expression, but in the presence of 5 mM ASA it was down-regulated, suggesting that nuclear factor kappaB (NF-kappaB) pathway can be involved in its regulation. PMID:19181355

  12. Proteomic analysis of nasal epithelial cells from cystic fibrosis patients.

    PubMed

    Jeanson, Ludovic; Guerrera, Ida Chiara; Papon, Jean-François; Chhuon, Cerina; Zadigue, Patricia; Prulière-Escabasse, Virginie; Amselem, Serge; Escudier, Estelle; Coste, André; Edelman, Aleksander

    2014-01-01

    The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

  13. Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells.

    PubMed

    Ogunwobi, Olorunseun Olatunji; Beales, Ian L P

    2006-05-15

    Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1beta-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-kappaB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-kappaB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.

  14. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    PubMed Central

    2009-01-01

    Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide. PMID:19650888

  15. Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis.

    PubMed Central

    Alderete, J F; Demeś, P; Gombosova, A; Valent, M; Fabusová, M; Jánoska, A; Stefanovic, J; Arroyo, R

    1988-01-01

    Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patients with trichomoniasis were purified, and VEC parasitism by Trichomonas vaginalis was examined. Trichomonads bound equally well to live or dead VECs, and up to 20% of VECs were parasitized. Trichomonal cytadherence of human VECs was time, temperature, and pH dependent. Saturation binding levels of live trichomonads to VECs gave approximately 2 organisms adherent to parasitized VEC. No differences in cytadherence levels were detected by different isolates to VECs from the same patient compared with adherence to VECs from normal individuals. Trypsinized, live T. vaginalis organisms failed to recognize VECs. A ligand assay identified four adhesin candidates, and only organisms without a prominent immunogen on the surface (negative phenotype) cytadhered to VECs and synthesized the adhesins, confirming the results of a recently published report by us on adherence to HeLa cell monolayers (J. F. Alderete and G. E. Garza, Infect. Immun. 56:28-33, 1988). These data show the ability of T. vaginalis to parasitize human vaginal epithelial cells in a specific receptor-ligand manner. PMID:3262088

  16. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

    PubMed

    Huang, Minghai; Wang, Bowen; Wan, Pengxia; Liang, Xuanwei; Wang, Xiaoran; Liu, Ying; Zhou, Qiang; Wang, Zhichong

    2015-02-01

    Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.

  17. K+ and Na+ absorption by outer sulcus epithelial cells.

    PubMed

    Marcus, D C; Chiba, T

    1999-08-01

    Transduction of sound into nerve impulses by hair cells depends on modulation of a current carried primarily by K+ into the cell across apical transduction channels that are permeable to cations. The cochlear function thus depends on active secretion of K+ accompanied by absorption of Na+ by epithelial cells enclosing the cochlear duct. The para-sensory cells which participate in the absorption of Na+ (down to the uniquely low level of 1 mM) were previously unidentified and the existence of a para-sensory pathway which actively absorbs K+ was previously unknown. A relative short circuit current (Isc,probe, measured as the extracellular current density with a vibrating electrode) was directed into the apical side of the outer sulcus epithelium, decreased by ouabain (1 mM), an inhibitor of Na+, K(+)-ATPase, and found to depend on bath Na+ and K+ but on neither Ca2+ nor Cl-. Isc,probe was shown to be an active current by its sensitivity to ouabain. On-cell patch clamp recordings of the apical membrane of outer sulcus cells displayed a channel activity, which carried inward currents under conditions identical to those used to measure Isc,probe. Both Isc,probe and non-selective cation channels (27.4+/-0.6 ps, n = 22) in excised outside-out patches from the apical membrane were inhibited by Gd3+ (1 mM). Ics,prob was also inhibited by 5 mM lidocaine, 1 mM quinine and 500 microM amiloride but not by 10 microM amiloride. These results demonstrate that outer sulcus epithelial cells contribute to the homeostasis of endolymph by actively absorbing Na+ and K+. An entry pathway in the apical membrane was shown to be through non-selective cation channels that were sensitive to Gd3+.

  18. Rac1 mediates intestinal epithelial cell apoptosis via JNK.

    PubMed

    Jin, Shi; Ray, Ramesh M; Johnson, Leonard R

    2006-12-01

    Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.

  19. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency. PMID:26673160

  20. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

  1. Hertwig's epithelial root sheath cell behavior during initial acellular cementogenesis in rat molars.

    PubMed

    Yamamoto, Tsuneyuki; Yamamoto, Tomomaya; Yamada, Tamaki; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

    2014-11-01

    This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig's epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial-mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle. PMID:24859538

  2. Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells

    SciTech Connect

    Oosthuizen, M.J.; Engels, F.; Van Esch, B.; Henricks, P.A.; Nijkamp, F.P. )

    1990-08-01

    Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

  3. Tumor cell-driven extracellular matrix remodeling drives haptotaxis during metastatic progression

    PubMed Central

    Oudin, Madeleine J.; Jonas, Oliver; Kosciuk, Tatsiana; Broye, Liliane C.; Guido, Bruna C.; Wyckoff, Jeff; Riquelme, Daisy; Lamar, John M.; Asokan, Sreeja B.; Whittaker, Charlie; Ma, Duanduan; Langer, Robert; Cima, Michael J.; Wisinski, Kari B.; Hynes, Richard O.; Lauffenburger, Douglas A.; Keely, Patricia J.; Bear, James E.; Gertler, Frank B.

    2016-01-01

    Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration. PMID:26811325

  4. FtsZ-independent septal recruitment and function of cell wall remodelling enzymes in chlamydial pathogens

    PubMed Central

    Frandi, Antonio; Jacquier, Nicolas; Théraulaz, Laurence; Greub, Gilbert; Viollier, Patrick H.

    2014-01-01

    The nature and assembly of the chlamydial division septum is poorly defined due to the paucity of a detectable peptidoglycan (PG)-based cell wall, the inhibition of constriction by penicillin and the presence of coding sequences for cell wall precursor and remodelling enzymes in the reduced chlamydial (pan-)genome. Here we show that the chlamydial amidase (AmiA) is active and remodels PG in Escherichia coli. Moreover, forward genetics using an E. coli amidase mutant as entry point reveals that the chlamydial LysM-domain protein NlpD is active in an E. coli reporter strain for PG endopeptidase activity (ΔnlpI). Immunolocalization unveils NlpD as the first septal (cell-wall-binding) protein in Chlamydiae and we show that its septal sequestration depends on prior cell wall synthesis. Since AmiA assembles into peripheral clusters, trimming of a PG-like polymer or precursors occurs throughout the chlamydial envelope, while NlpD targets PG-like peptide crosslinks at the chlamydial septum during constriction. PMID:24953095

  5. FtsZ-independent septal recruitment and function of cell wall remodelling enzymes in chlamydial pathogens.

    PubMed

    Frandi, Antonio; Jacquier, Nicolas; Théraulaz, Laurence; Greub, Gilbert; Viollier, Patrick H

    2014-06-23

    The nature and assembly of the chlamydial division septum is poorly defined due to the paucity of a detectable peptidoglycan (PG)-based cell wall, the inhibition of constriction by penicillin and the presence of coding sequences for cell wall precursor and remodelling enzymes in the reduced chlamydial (pan-)genome. Here we show that the chlamydial amidase (AmiA) is active and remodels PG in Escherichia coli. Moreover, forward genetics using an E. coli amidase mutant as entry point reveals that the chlamydial LysM-domain protein NlpD is active in an E. coli reporter strain for PG endopeptidase activity (ΔnlpI). Immunolocalization unveils NlpD as the first septal (cell-wall-binding) protein in Chlamydiae and we show that its septal sequestration depends on prior cell wall synthesis. Since AmiA assembles into peripheral clusters, trimming of a PG-like polymer or precursors occurs throughout the chlamydial envelope, while NlpD targets PG-like peptide crosslinks at the chlamydial septum during constriction.

  6. The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells.

    PubMed

    Jolly, Carrie; Winfree, Seth; Hansen, Bryan; Steele-Mortimer, Olivia

    2014-01-01

    The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non-phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin-driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium- and phospholipid-binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS-mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho-family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.

  7. Growth of respiratory syncytial virus in mink lung epithelial cells.

    PubMed

    Yeolekar, L R; Damle, R G; Basu, A; Rao, B L

    2002-12-01

    Mink lung epithelial cells (Mv-1-Lu) were tested for their ability to support the growth and serial passage of respiratory syncytial virus (RSV) in vitro. Indian isolates of RSV induced distinctive cytopathic effect with typical rounding of cells followed by detachment with more than 50 per cent cells showing bright fluorescence using anti-RSV monoclonal antibodies in immunofluorescence test. Serial passage of RSV was possible in Mv-1-Lu cells without loss of sensitivity of the cells for virus growth. Titration of cell associated virus and virus released in the supernatant indicated that 60 per cent of the virus was released in the supernatant, and 40 per cent remained cell associated. Transmission electron microscopic studies of negatively stained RSV particles and ultra-thin sections of RSV infected Mv-1-Lu cells showed roughly spherical particles with club shaped projections, budding from the cytoplasmic membrane. These results indicate that Mv-1-Lu cell line is suitable for the growth and propagation of RSV.

  8. Effect of curcumin on aging retinal pigment epithelial cells.

    PubMed

    Zhu, Wei; Wu, Yan; Meng, Yi-Fang; Wang, Jin-Yu; Xu, Ming; Tao, Jian-Jun; Lu, Jiong

    2015-01-01

    Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE