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Sample records for remodeling epithelial cell

  1. Epithelial Cell Proliferation Contributes to Airway Remodeling in Severe Asthma

    PubMed Central

    Cohen, Lance; E, Xueping; Tarsi, Jaime; Ramkumar, Thiruvamoor; Horiuchi, Todd K.; Cochran, Rebecca; DeMartino, Steve; Schechtman, Kenneth B.; Hussain, Iftikhar; Holtzman, Michael J.; Castro, Mario

    2007-01-01

    Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. Measurements and Main Results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p = 0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p = 0.002) and Ki67 was increased (p < 0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p < 0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p = 0.002), suggesting increased cell death. Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe

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

    PubMed

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

    2016-07-19

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

  3. 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

  4. Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis.

    PubMed

    Higashi, Tomohito; Arnold, Torey R; Stephenson, Rachel E; Dinshaw, Kayla M; Miller, Ann L

    2016-07-25

    Epithelial integrity and barrier function must be maintained during the complex cell shape changes that occur during cytokinesis in vertebrate epithelial tissue. Here, we investigate how adherens junctions and bicellular and tricellular tight junctions are maintained and remodeled during cell division in the Xenopus laevis embryo. We find that epithelial barrier function is not disrupted during cytokinesis and is mediated by sustained tight junctions. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that adherens junction proteins are stabilized at the cleavage furrow by increased tension. We find that Vinculin is recruited to the adherens junction at the cleavage furrow, and that inhibiting recruitment of Vinculin by expressing a dominant-negative mutant increases the rate of furrow ingression. Furthermore, we show that cells neighboring the cleavage plane are pulled between the daughter cells, making a new interface between neighbors, and two new tricellular tight junctions flank the midbody following cytokinesis. Our data provide new insight into how epithelial integrity and barrier function are maintained throughout cytokinesis in vertebrate epithelial tissue. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm.

    PubMed

    Seifert, Jessica R K; Lehmann, Ruth

    2012-06-01

    Trans-epithelial migration describes the ability of migrating cells to cross epithelial tissues and occurs during development, infection, inflammation, immune surveillance, wound healing and cancer metastasis. Here we investigate Drosophila primordial germ cells (PGCs), which migrate through the endodermal epithelium. Through live imaging and genetic experimentation we demonstrate that PGCs take advantage of endodermal tissue remodeling to gain access to the gonadal mesoderm and are unable to migrate through intact epithelial tissues. These results are in contrast to the behavior of leukocytes, which actively loosen epithelial junctions to migrate, and raise the possibility that in other contexts in which migrating cells appear to breach tissue barriers, they are actually exploiting existing tissue permeability. Therefore, the use of active invasive programs is not the sole mechanism to infiltrate tissues.

  6. Role of Porphyromonas gingivalis SerB in gingival epithelial cell cytoskeletal remodeling and cytokine production.

    PubMed

    Hasegawa, Yoshiaki; Tribble, Gena D; Baker, Henry V; Mans, Jeffrey J; Handfield, Martin; Lamont, Richard J

    2008-06-01

    The SerB protein of Porphyromonas gingivalis is a HAD family serine phosphatase that plays a critical role in entry and survival of the organism in gingival epithelial cells. SerB is secreted by P. gingivalis upon contact with epithelial cells. Here it is shown by microarray analysis that SerB impacts the transcriptional profile of gingival epithelial cells, with pathways involving the actin cytoskeleton and cytokine production among those significantly overpopulated with differentially regulated genes. Consistent with the transcriptional profile, a SerB mutant of P. gingivalis exhibited defective remodeling of actin in epithelial cells. Interaction between gingival epithelial cells and isolated SerB protein resulted in actin rearrangement and an increase in the F/G actin ratio. SerB protein was also required for P. gingivalis to antagonize interleukin-8 accumulation following stimulation of epithelial cells with Fusobacterium nucleatum. SerB is thus capable of modulating host cell signal transduction that impacts the actin cytoskeleton and cytokine production.

  7. Role of Porphyromonas gingivalis SerB in Gingival Epithelial Cell Cytoskeletal Remodeling and Cytokine Production▿

    PubMed Central

    Hasegawa, Yoshiaki; Tribble, Gena D.; Baker, Henry V.; Mans, Jeffrey J.; Handfield, Martin; Lamont, Richard J.

    2008-01-01

    The SerB protein of Porphyromonas gingivalis is a HAD family serine phosphatase that plays a critical role in entry and survival of the organism in gingival epithelial cells. SerB is secreted by P. gingivalis upon contact with epithelial cells. Here it is shown by microarray analysis that SerB impacts the transcriptional profile of gingival epithelial cells, with pathways involving the actin cytoskeleton and cytokine production among those significantly overpopulated with differentially regulated genes. Consistent with the transcriptional profile, a SerB mutant of P. gingivalis exhibited defective remodeling of actin in epithelial cells. Interaction between gingival epithelial cells and isolated SerB protein resulted in actin rearrangement and an increase in the F/G actin ratio. SerB protein was also required for P. gingivalis to antagonize interleukin-8 accumulation following stimulation of epithelial cells with Fusobacterium nucleatum. SerB is thus capable of modulating host cell signal transduction that impacts the actin cytoskeleton and cytokine production. PMID:18391005

  8. Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

    PubMed Central

    Rother, Jan; Büchsenschütz-Göbeler, Matthias; Nöding, Helen; Steltenkamp, Siegfried; Samwer, Konrad; Janshoff, Andreas

    2015-01-01

    The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m−1, and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells. PMID:25566882

  9. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

    PubMed

    Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

    2015-02-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The

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

    PubMed

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

    2016-09-08

    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 TRF1(fl/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 TRF1(fl/fl) mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction.

  11. 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

  12. 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

  13. 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. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2017-01-01

    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

  15. 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

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

    PubMed

    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; van der Vliet, Albert

    2016-11-03

    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.

  17. 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.

  18. The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures.

    PubMed

    Nezi, Luigi; Greco, Dario; Nitsch, Lucio; Garbi, Corrado

    2002-01-01

    Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts. FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.

  19. 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.

  20. 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

  1. 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.

  2. KIF17 regulates RhoA-dependent actin remodeling at epithelial cell–cell adhesions

    PubMed Central

    Acharya, Bipul R.; Espenel, Cedric; Libanje, Fotine; Raingeaud, Joel; Morgan, Jessica; Jaulin, Fanny; Kreitzer, Geri

    2016-01-01

    ABSTRACT The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell–cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP–actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell–cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilinS3A inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA–GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells. PMID:26759174

  3. STIM1 accelerates cell senescence in a remodeled microenvironment but enhances the epithelial-to-mesenchymal transition in prostate cancer

    PubMed Central

    Xu, Yingxi; Zhang, Shu; Niu, Haiying; Ye, Yujie; Hu, Fen; Chen, Si; Li, Xuefei; Luo, Xiaohe; Jiang, Shan; Liu, Yanhua; Chen, Yanan; Li, Junying; Xiang, Rong; Li, Na

    2015-01-01

    The importance of store-operated Ca2+ entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging. Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells. In addition, STIM1 and ORAI1 inhibited NF-κB signaling and remodeled the tumor microenvironment by reducing the formation of M2 phenotype macrophages, possibly creating an unfavorable tumor microenvironment and inhibiting cancer development. However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways. Thus, our study revealed novel regulatory effects and the mechanisms by which STIM1 affects cell senescence, tumor migration and the tumor microenvironment, revealing that STIM1 has multiple functions in prostate cancer cells. PMID:26257076

  4. STIM1 accelerates cell senescence in a remodeled microenvironment but enhances the epithelial-to-mesenchymal transition in prostate cancer.

    PubMed

    Xu, Yingxi; Zhang, Shu; Niu, Haiying; Ye, Yujie; Hu, Fen; Chen, Si; Li, Xuefei; Luo, Xiaohe; Jiang, Shan; Liu, Yanhua; Chen, Yanan; Li, Junying; Xiang, Rong; Li, Na

    2015-08-10

    The importance of store-operated Ca(2+) entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging. Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells. In addition, STIM1 and ORAI1 inhibited NF-κB signaling and remodeled the tumor microenvironment by reducing the formation of M2 phenotype macrophages, possibly creating an unfavorable tumor microenvironment and inhibiting cancer development. However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways. Thus, our study revealed novel regulatory effects and the mechanisms by which STIM1 affects cell senescence, tumor migration and the tumor microenvironment, revealing that STIM1 has multiple functions in prostate cancer cells.

  5. HIF-1alpha regulates epithelial inflammation by cell autonomous NFkappaB activation and paracrine stromal remodeling.

    PubMed

    Scortegagna, Marzia; Cataisson, Christophe; Martin, Rebecca J; Hicklin, Daniel J; Schreiber, Robert D; Yuspa, Stuart H; Arbeit, Jeffrey M

    2008-04-01

    Hypoxia inducible factor-1 (HIF-1) is a master regulatory transcription factor controlling multiple cell-autonomous and non-cell-autonomous processes, such as metabolism, angiogenesis, matrix invasion, and cancer metastasis. Here we used a new line of transgenic mice with constitutive gain of HIF-1 function in basal keratinocytes and demonstrated a signaling pathway from HIF-1 to nuclear factor kappa B (NFkappaB) activation to enhanced epithelial chemokine and cytokine elaboration. This pathway was responsible for a phenotypically silent accumulation of stromal inflammatory cells and a marked inflammatory hypersensitivity to a single 12-O-tetradecanoylphorbol-13-acetate (TPA) challenge. HIF-1-induced NFkappaB activation was composed of 2 elements, IkappaB hyperphosphorylation and phosphorylation of Ser276 on p65, enhancing p65 nuclear localization and transcriptional activity, respectively. NFkappaB transcriptional targets macrophage inflammatory protein-2 (MIP-2/CXCL2/3), keratinocyte chemokine (KC/CXCL1), and tumor necrosis factor [alfa] (TNFalpha) were constitutively up-regulated and further increased after TPA challenge both in cultured keratinocytes and in transgenic mice. Whole animal KC, MIP-2, or TNFalpha immunodepletion each abrogated TPA-induced inflammation, whereas blockade of either VEGF or placenta growth factor (PlGF) signaling did not affect transgenic inflammatory hyper-responsiveness. Thus, epithelial HIF-1 gain of function remodels the local environment by cell-autonomous NFkappaB-mediated chemokine and cytokine secretion, which may be another mechanism by which HIF-1 facilitates either inflammatory diseases or malignant progression.

  6. Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function.

    PubMed

    Cook, Christi D; Hill, Abby S; Guo, Margaret; Stockdale, Linda; Papps, Julia P; Isaacson, Keith B; Lauffenburger, Douglas A; Griffith, Linda G

    2017-04-18

    Mucosal barrier tissues, comprising a layer of tightly-bonded epithelial cells in intimate molecular communication with an underlying matrix-rich stroma containing fibroblasts and immune cells, are prominent targets for drugs against infection, chronic inflammation, and other disease processes. Although human in vitro models of such barriers are needed for mechanistic studies and drug development, differences in extracellular matrix (ECM) needs of epithelial and stromal cells hinder efforts to create such models. Here, using the endometrium as an example mucosal barrier, we describe a synthetic, modular ECM hydrogel suitable for 3D functional co-culture, featuring components that can be remodeled by cells and that respond dynamically to sequester local cell-secreted ECM characteristic of each cell type. The synthetic hydrogel combines peptides with off-the-shelf reagents and is thus accessible to cell biology labs. Specifically, we first identified a single peptide as suitable for initial attachment of both endometrial epithelial and stromal cells using a 2D semi-empirical screen. Then, using a co-culture system of epithelial cells cultured on top of gel-encapsulated stromal cells, we show that inclusion of ECM-binding peptides in the hydrogel, along with the integrin-binding peptide, leads to enhanced accumulation of basement membrane beneath the epithelial layer and more fibrillar collagen matrix assembly by stromal cells over two weeks in culture. Importantly, endometrial co-cultures composed of either cell lines or primary cells displayed hormone-mediated differentiation as assessed by morphological changes and secretory protein production. A multiplex analysis of apical cytokine and growth factor secretion comparing cell lines and primary cells revealed strikingly different patterns, underscoring the importance of using primary cell models in analysis of cell-cell communication networks. In summary, we define a "one-size-fits-all" synthetic ECM that enables

  7. IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: Effect of Tiotropium.

    PubMed

    Anzalone, Giulia; Gagliardo, Rosalia; Bucchieri, Fabio; Albano, Giusy Daniela; Siena, Liboria; Montalbano, Angela Marina; Bonanno, Anna; Riccobono, Loredana; Pieper, Michael Paul; Gjomarkaj, Mark; Profita, Mirella

    2016-05-01

    IL-17A plays a key role in the persistence of airway inflammation, oxidative stress, and reduction of steroid-sensitivity in COPD. We studied the effect of IL-17A on chromatin remodeling and IL-8 production. We measured the levels of IL-8 and IL-17A in induced sputum supernatants (ISS) from healthy controls (HCs), healthy smokers (HSs), and COPD patients by enzyme-linked immunosorbent assay (ELISA). A human bronchial epithelial cell line (16HBE) was stimulated with ISS from HCs, HSs, or COPD subjects. IL-8 was evaluated in 16HBE by Western blot and real-time polymerase chain reaction (PCR). Histone deacetylase 2 (HDAC2), acetyl histone H3 (Ac-His H3) (k9) and inhibitor kappa kinase alpha (IKKα) levels were evaluated in the nuclear extract by Western blot. Finally, we evaluated the effect of IL-17A depletion in ISS, the silencing of IKKα, and the anti-inflammatory effects of Tiotropium Spiriva® (100nM) on 16HBE. IL-8 and IL-17A levels were higher in ISS from COPD patients and HSs than from HCs. IL-8 protein and messenger RNA (mRNA) levels were increased in 16HBE stimulated with ISS from COPD patients compared with untreated cells. Furthermore, ISS from COPD patients reduced the nuclear levels of HDAC2 while increasing the activity of both Ac-His H3 (k9) and IKKα in stimulated 16HBE. IL-17A depletion in ISS and the IKKα silencing in 16HBE significantly increased the nuclear levels of HDAC2, reduced Ac-His H3 (k9), and promoted IL-8 synthesis in stimulated 16HBE. Tiotropium controls the proinflammatory activity generated by ISS from COPD patients in 16HBE. IL-17A present in the airway of COPD patients, which induces chromatin remodeling, promotes the release of IL-8 in the bronchial epithelium. Tiotropium is able to control this proinflammatory activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Airborne particulate matter in vitro exposure induces cytoskeleton remodeling through activation of the ROCK-MYPT1-MLC pathway in A549 epithelial lung cells.

    PubMed

    Chirino, Yolanda I; García-Cuellar, Claudia María; García-García, Carlos; Soto-Reyes, Ernesto; Osornio-Vargas, Álvaro Román; Herrera, Luis A; López-Saavedra, Alejandro; Miranda, Javier; Quintana-Belmares, Raúl; Pérez, Irma Rosas; Sánchez-Pérez, Yesennia

    2017-03-06

    Airborne particulate matter with an aerodynamic diameter ≤10μm (PM10) is considered a risk factor for the development of lung cancer. Little is known about the cellular mechanisms by which PM10 is associated with cancer, but there is evidence that its exposure can lead to an acquired invasive phenotype, apoptosis evasion, inflammasome activation, and cytoskeleton remodeling in lung epithelial cells. Cytoskeleton remodeling occurs through actin stress fiber formation, which is partially regulated through ROCK kinase activation, we aimed to investigate if this protein was activated in response to PM10 exposure in A549 lung epithelial cells. Results showed that 10μg/cm(2) of PM10 had no influence on cell viability but increased actin stress fibers, cytoplasmic ROCK expression, and phosphorylation of myosin phosphatase-targeting 1 (MYPT1) and myosin light chain (MLC) proteins, which are targeted by ROCK. The inhibition of ROCK prevented actin stress fiber formation and the phosphorylation of MYPT1 and MLC, suggesting that PM10 activated the ROCK-MYPT1-MLC pathway in lung epithelial cells. The activation of ROCK1 has been involved in the acquisition of malignant phenotypes, and its induction by PM10 exposure could contribute to the understanding of PM10 as a risk factor for cancer development through the mechanisms associated with invasive phenotype.

  9. Remodeling of the tight junction during recovery from exposure to hydrogen peroxide in kidney epithelial cells

    PubMed Central

    Gonzalez, Jeannette E.; DiGeronimo, Robert J.; Arthur, D’Ann E.; King, Jonathan M.

    2009-01-01

    Renal ischemia-reperfusion injury results in oxidative stress-induced alterations in barrier function. Activation of the mitogen-activated protein (MAP) kinase pathway during recovery from oxidative stress may be an effector of oxidant-induced tight junction reorganization. We hypothesized that tight junction composition and barrier function would be perturbed during recovery from oxidative stress. We developed a model of short-term H2O2 exposure followed by recovery using Madin Darby Canine Kidney cells (MDCK II). H2O2 perturbs barrier function without a significant cytotoxic effect except in significant doses. ERK-1/2 and p38, both enzymes of the MAP kinase pathway, were activated within minutes of exposure to H2O2. Transient exposure to H2O2 produced a biphasic response in transepithelial electrical resistance (TER). An initial drop in TER at 6 hours was followed by a significant increase at 24 hours. Inhibition of ERK-1/2 activation attenuated the increase in TER observed at 24 hours. Expression of occludin initially decreased followed by partial recovery at 24 hours. In contrast, claudin-1 levels decreased and failed to recover at 24 hours. Claudin-2 levels markedly decreased at 24 hours; however, inhibition of ERK-1/2 activation was protective. Occludin and claudin-1 localization at the apical membrane on immunofluorescent images was fragmented at 6 hours after H2O2 exposure with subsequent recovery of appropriate localization by 24 hours. MDCK II cell recovery after H2O2 exposure is associated with functional and structural modification of the tight junction that are mediated in part by activation of the MAP kinase enzymes, ERK-1/2 and p38. PMID:19733232

  10. 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

  11. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2016-02-08

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

  14. Semen modulated secretory activity of oviductal epithelial cells is linked to cellular proteostasis network remodeling: Proteomic insights into the early phase of interaction in the oviduct in vivo.

    PubMed

    Steinberger, Birgit; Yu, Hans; Brodmann, Theodor; Milovanovic, Daniela; Reichart, Ursula; Besenfelder, Urban; Artemenko, Konstantin; Razzazi-Fazeli, Ebrahim; Brem, Gottfried; Mayrhofer, Corina

    2017-06-23

    interest, also with regard to in vitro purposes. So far, the role of the early phase of interaction in the female organ has not been considered in detail. To get a further insight into the underlying cellular and molecular mechanisms, herein we analyzed the effect of semen on oviductal epithelial cells (Oecs) on the intracellular proteome level within the first two hours after insemination. The present study revealed a directed response of Oecs in vivo and disclosed intracellular pathways that are affected by the interplay between semen and the female reproductive tract. The prompt adaptation of the secretory activity and remodeling of the oviductal epithelium was accompanied by the concerted alterations of protein species that are primarily involved in the maintenance of cellular homeostasis. Besides emphasizing the importance of the early interaction phase for subsequent reproductive processes, the gained knowledge might further be implemented for in vitro applications as well. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Nasal epithelial repair and remodeling in physical injury, infection, and inflammatory diseases.

    PubMed

    Yan, Yan; Gordon, William M; Wang, De-Yun

    2013-06-01

    To summarize the current knowledge of cellular and molecular mechanisms of nasal epithelial repair and remodeling during physical and pathophysiological conditions. Nasal epithelial repair and remodeling is a highly organized and well coordinated process, involving inflammation, proliferation, differentiation, matrix deposition, and remodeling, and is regulated by a wide variety of growth factors and cytokines. From the in-vivo and in-vitro studies conducted in both human and animal models, undifferentiated basal cells (progenitors) are able to migrate from adjacent epithelium, spread over the denuded basement membrane, and proliferate in injured regions (self-renewal) in necessary (homeostasis) or excessive (hyperplasia) degree. Progenitor cells reorient to an apical-basal polarity, and progressively differentiate into ciliated and nonciliated columnar cells and goblet cells, reconstituting a functional respiratory epithelium after several weeks. This recovery process can be observed during various types and severity of injury, and also in common nasal diseases, including acute viral, allergic, and nonallergic rhinitis, as well as chronic rhinosinusitis with and without nasal polyps. Although nearly 10 000 articles about nasal epithelium have been published in the last decade, the mechanisms underlying the nasal epithelial repair are still understood at only a superficial descriptive level. In order to advance rhinology to the next level of a comprehensive knowledge of the orchestrated genetic and molecular processes acting during epithelial repair, combined clinical and experimental studies using sophisticated investigational plans to elucidate the functions of both the protein-coding and regulatory portions of the human genome are required.

  16. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression.

    PubMed

    Haynes, Jennifer; Srivastava, Jyoti; Madson, Nikki; Wittmann, Torsten; Barber, Diane L

    2011-12-01

    Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.

  17. Autophagy plays a role in FSTL1-induced epithelial mesenchymal transition and airway remodeling in asthma.

    PubMed

    Liu, Tian; Liu, Yahui; Miller, Marina; Cao, Liuzhao; Zhao, Jiping; Wu, Jinxiang; Wang, Junfei; Liu, Lin; Li, Shuo; Zou, Minfang; Xu, Jiawei; Broide, David H; Dong, Liang

    2017-07-01

    Asthma is a chronic disease related to airway hyperresponsiveness and airway remodeling. Airway remodeling is the important reason of refractory asthma and is associated with differentiation of airway epithelia into myofibroblasts via epithelial-mesenchymal transition (EMT) to increase the process of subepithelial fibrosis. There is growing evidence that autophagy modulates remodeling. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we hypothesized that Follistatin-like 1 (FSTL1) promotes EMT and airway remodeling by intensifying autophagy. With the use of transmission electron microscopy (TEM), double-membrane autophagosomes were detected in the airways of patients and mice. More autophagosomes were in patients with asthma and OVA-challenged mice compared with healthy controls. The expression of FSTL1 and beclin-1 was upregulated in the airways of patients with asthma and OVA-challenged mice, accompanied by airway EMT and remodeling. In OVA-challenged Fstl1(+/-) mice, the degree of airway remodeling and autophagy was decreased compared with control mice. The effects of FSTL1 on autophagy and EMT were also tested in 16HBE cells in vitro. Additionally, inhibition of autophagy by using LY-294002 and siRNA-ATG5 reduced the FSTL1-induced EMT in 16HBE cells, as measured by E-cadherin, N-cadherin, and vimentin expression. In line herewith, administration of LY-294002 reduced the expression of autophagy, EMT, and airway remodeling markers in FSTL1-challenged WT mice. Taken together, our study suggests that FSTL1 may induce EMT and airway remodeling by activating autophagy. These findings may provide novel avenues for therapeutic research targeting the autophagy and FSTL1 pathway, which may be beneficial to patients with refractory asthma. Copyright © 2017 the American Physiological Society.

  18. Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease.

    PubMed

    Puchelle, Edith; Zahm, Jean-Marie; Tournier, Jean-Marie; Coraux, Christelle

    2006-11-01

    In chronic obstructive pulmonary disease (COPD), exacerbations are generally associated with several causes, including pollutants, viruses, bacteria that are responsible for an excess of inflammatory mediators, and proinflammatory cytokines released by activated epithelial and inflammatory cells. The normal response of the airway surface epithelium to injury includes a succession of cellular events, varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even complete denudation of the basement membrane. The epithelium then has to repair and regenerate to restore its functions, through several mechanisms, including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. In COPD, the remodeling of the airway epithelium, such as squamous metaplasia and mucous hyperplasia that occur during injury, may considerably disturb the innate immune functions of the airway epithelium. In vitro and in vivo models of airway epithelial wound repair and regeneration allow the study of the spatiotemporal modulation of cellular and molecular interaction factors-namely, the proinflammatory cytokines, the matrix metalloproteinases and their inhibitors, and the intercellular adhesion molecules. These factors may be markedly altered during exacerbation periods of COPD and their dysregulation may induce remodeling of the airway mucosa and a leakiness of the airway surface epithelium. More knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to cytoprotective and regenerative therapeutics, allowing the reconstitution of a functional, well-differentiated airway epithelium in COPD.

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

    PubMed

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

    2008-08-15

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

  20. Systems biology approaches to understanding Epithelial Mesenchymal Transition (EMT) in mucosal remodeling and signaling in asthma

    PubMed Central

    2014-01-01

    A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma. PMID:24982697

  1. 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

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

    PubMed

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

    2010-08-01

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

  3. 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

    2015-11-05

    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

  4. Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

    PubMed

    Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

    2016-11-01

    Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

  5. Oxidative damage and TGF-β differentially induce lung epithelial cell sonic hedgehog and tenascin-C expression: implications for the regulation of lung remodelling in idiopathic interstitial lung disease

    PubMed Central

    Fitch, Paul M; Howie, Sarah E M; Wallace, William A H

    2011-01-01

    Idiopathic interstitial lung diseases (iILDs) are characterized by inflammation, hyperplasia of Type-II alveolar epithelial cells (AECs) and lung remodelling often with progressive fibrosis. It remains unclear which signals initiate iILD and/or maintain the disease processes. Using real-time RT-PCR and immunohistochemistry on archival biopsies of three patterns of iILD (usual interstitial pneumonitis/UIP, non-specific interstitial pneumonitis/NSIP and cryptogenic organizing pneumonia/COP) we investigated whether hedgehog signalling (previously associated with lung damage and repair) was functional and whether the damage associated extracellular matrix protein tenascin-C was present in activated Type-II AECs in all three iILDs. Using tissue culture, protein and mRNA detection we also determined how two signals (oxidative damage and TGF-β) associated with iILD pathogenesis affected Sonic hedgehog (SHH) and tenascin-C production by a Type-II AEC cell line. We report that SHH pathway and tenascin-C mRNA and proteins were found in UIP, NSIP and COP. SHH signalling was most active at sites of immature organizing fibrous tissue (fibroblastic foci) in UIP. In vitro Type-II AECs constitutively secrete SHH but not tenascin-C. Oxidative injury stimulated SHH release whereas TGF-β inhibited it. TGF-β and oxidative damage both upregulated tenascin-C mRNA but only TGF-β induced synthesis and release of a distinct protein isoform. SHH signalling is active in Type-II AECs from three types of ILD and all three express tenascin-C. PMID:21039988

  6. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    PubMed

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

  7. Lung Epithelial Progenitor Cells

    PubMed Central

    Rawlins, Emma L.

    2008-01-01

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

  8. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration.

    PubMed

    Kumar, J Dinesh; Steele, Islay; Moore, Andrew R; Murugesan, Senthil V; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea; Dockray, Graham J

    2015-07-15

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling.

  9. 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.

  10. Integrins and epithelial cell polarity.

    PubMed

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

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

  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. PDK1 Regulates Vascular Remodeling and Promotes Epithelial-Mesenchymal Transition in Cardiac Development▿

    PubMed Central

    Feng, Qiuting; Di, Ruomin; Tao, Fang; Chang, Zai; Lu, Shuangshuang; Fan, Wenjing; Shan, Congjia; Li, Xinli; Yang, Zhongzhou

    2010-01-01

    One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail. PMID:20457809

  13. Endothelial cell dynamics in vascular remodelling.

    PubMed

    Barbacena, Pedro; Carvalho, Joana R; Franco, Claudio A

    2016-01-01

    In this ESCHM 2016 conference talk report, we summarise two recently published original articles Franco et al. PLoS Biology 2015 and Franco et al. eLIFE 2016. The vascular network undergoes extensive vessel remodelling to become fully functional. Is it well established that blood flow is a main driver for vascular remodelling. It has also been proposed that vessel pruning is a central process within physiological vessel remodelling. However, despite its central function, the cellular and molecular mechanisms regulating vessel regression, and their interaction with blood flow patterns, remain largely unexplained. We investigated the cellular process governing developmental vascular remodelling in mouse and zebrafish. We established that polarised reorganization of endothelial cells is at the core of vessel regression, representing vessel anastomosis in reverse. Moreover, we established for the first time an axial polarity map for all endothelial cells together with an in silico method for the computation of the haemodynamic forces in the murine retinal vasculature. Using network-level analysis and microfluidics, we showed that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/11 renders endothelial cells more sensitive to shear, resulting in axial polarisation at lower shear stress levels. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.

  14. 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

  15. The SYT-SSX fusion protein and histological epithelial differentiation in synovial sarcoma: relationship with extracellular matrix remodeling.

    PubMed

    Saito, Tsuyoshi

    2013-01-01

    Synovial sarcoma (SS) tumor cells, which have the chromosomal translocation t(X;18)(p11.2;q11.2), have an inherently greater propensity for epithelial differentiation than other mesenchymal tumors, especially spindle cell sarcomas. This is caused by de-repression of the transcription of E-cadherin by SYT-SSX1 and SYT-SSX2, which dissociate Snail or Slug, respectively, from the E-cadherin promoter. However, a subset of SS with SYT-SSX1 loses E-cadherin expression despite adequate de-repression because of mutations in E-cadherin, resulting in monophasic histology. The ratio of the expression levels of SYT-SSX1 and Snail is also associated with E-cadherin expression: the lower the SYT-SSX1/Snail ratio, the lower the level of E-cadherin expression, and vice versa, thus affecting tumor histology. In addition, Wnt signal activation caused by mutation of β-catenin, APC, or Axin 1 and 2 is associated with monophasic histology. Remodeling of the extracellular matrix is also important. Only cells that survive all of these steps can finally exhibit biphasic histology. On the other hand, the SYT-SSX2 fusion has a weaker de-repression effect on the E-cadherin promoter than does SYT-SSX1, so it is difficult for SYT-SSX2-expressing tumors to achieve sufficient capacity for epithelial differentiation to form glandular structures. This review provides an interesting model for this epithelial differentiation that shows a possible mechanism for the aberrant mesenchymal to epithelial transition of SS and suggests that it might better be considered an epithelial to mesenchymal transition.

  16. 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

  17. 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.

  18. Targeting granulocyte-macrophage colony-stimulating factor in epithelial and vascular remodeling in experimental eosinophilic esophagitis.

    PubMed

    McNamee, E N; Biette, K A; Hammer, J; Harris, R; Miyazawa, H; Lee, J J; Furuta, G T; Masterson, J C

    2017-08-01

    Eosinophilic esophagitis (EoE) is a chronic antigen-mediated clinicopathologic disease of the esophagus characterized by an eosinophil-predominant inflammatory infiltrate. A clinical hallmark is extensive tissue remodeling including basal zone hyperplasia, fibrosis, and angiogenesis. However, the cellular mechanisms responsible for these processes are not fully defined. We hypothesized that targeting granulocyte-macrophage colony-stimulating factor (GM-CSF; an agonist cytokine linked with eosinophil survival and activation) would be protective in a preclinical model of EoE. Eosinophilic esophagitis-like esophageal inflammation was induced in the L2-IL5(OXA) EoE mouse model, and GM-CSF production was assessed by mRNA and protein analyses. Granulocyte-macrophage colony-stimulating factor-receptor-alpha expression patterns were examined by flow cytometric and immunofluorescence analysis. L2-IL5(OXA) EoE mice were treated with anti-GM-CSF neutralizing antibody or isotype control and assessed for histopathological indices of eosinophilia, epithelial hyperplasia, and angiogenesis by immunohistochemistry and RT-PCR. Significantly increased levels of esophageal GM-CSF expression was detected in the L2-IL5(OXA) mouse EoE model during active inflammation. Granulocyte-macrophage colony-stimulating factor-receptor-alpha was predominantly expressed on esophageal eosinophils during EoE, in addition to select cells within the lamina propria. Anti-GM-CSF neutralization in L2-IL5(OXA) EoE mice resulted in a significant diminution of epithelial eosinophilia in addition to basal cell hyperplasia and vascular remodeling. This treatment response was independent of effects on esophageal eosinophil maturation or activation. Granulocyte-macrophage colony-stimulating factor is a potential therapeutic target to reduce esophageal eosinophilia and remodeling. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. 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.

  20. Remodeling of the epithelial-connective tissue interface (ECTI) in oral epithelial dysplasia as visualized by noninvasive 3D imaging

    PubMed Central

    Pal, Rahul; Shilagard, Tuya; Yang, Jinping; Villarreal, Paula; Brown, Tyra; Qiu, Suimin; McCammon, Susan; Resto, Vicente; Vargas, Gracie

    2016-01-01

    Early neoplastic features in oral epithelial dysplasia are first evident at the basal epithelium positioned at the epithelial-connective tissue interface (ECTI), separating the basal epithelium from the underlying lamina propria. The ECTI undergoes significant deformation in early neoplasia due to focal epithelial expansion and proteolytic remodeling of the lamina propria but few studies have examined these changes. In the present study, we quantitated alterations in ECTI topography in dysplasia using in vivo volumetric multiphoton autofluorescence microscopy and second harmonic generation microscopy. The label-free method allows direct noninvasive visualization of the ECTI surface without perturbing the epithelium. An image-based parameter, ‘ECTI contour’, is described that indicates deformation of the ECTI surface. ECTI contour was higher in dysplasia than control or inflammed specimens, indicating transition from flat to a deformed surface. Cellular parameters of nuclear area, nuclear density, coefficient of variation in nuclear area in the basal epithelium and collagen density in areas adjacent to ECTI were measured. ECTI contour differentiated dysplasia from control/benign mucosa with higher sensitivity and specificity than basal nuclear density or basal nuclear area, comparable to coefficient of variation in nuclear area and collagen density. The presented method offers a unique opportunity to study ECTI in intact mucosa with simultaneous assessment of cellular and extracellular matrix features, expanding opportunities for studies of early neoplastic events near this critical interface and potentially leading to development of new approaches for detecting neoplasia in vivo. PMID:27302162

  1. Glioma progression through the prism of heat shock protein mediated extracellular matrix remodeling and epithelial to mesenchymal transition.

    PubMed

    Rajesh, Y; Biswas, Angana; Mandal, Mahitosh

    2017-10-15

    Glial tumor is one of the intrinsic brain tumors with high migratory and infiltrative potential. This essentially contributes to the overall poor prognosis by circumvention of conventional treatment regimen in glioma. The underlying mechanism in gliomagenesis is bestowed by two processes- Extracellular matrix (ECM) Remodeling and Epithelial to mesenchymal transition (EMT). Heat Shock Family of proteins (HSPs), commonly known as "molecular chaperons" are documented to be upregulated in glioma. A positive correlation also exists between elevated expression of HSPs and invasive capacity of glial tumor. HSPs overexpression leads to mutational changes in glioma, which ultimately drive cells towards EMT, ECM modification, malignancy and invasion. Differential expression of HSPs - a factor providing cytoprotection to glioma cells, also contributes towards its radioresistance /chemoresistance. Various evidences also display upregulation of EMT and ECM markers by various heat shock inducing proteins e.g. HSF-1. The aim of this review is to study in detail the role of HSPs in EMT and ECM leading to radioresistance/chemoresistance of glioma cells. The existing treatment regimen for glioma could be enhanced by targeting HSPs through immunotherapy, miRNA and exosome mediated strategies. This could be envisaged by better understanding of molecular mechanisms underlying glial tumorigenesis in relation to EMT and ECM remodeling under HSPs influence. Our review might showcase fresh potential for the development of next generation therapeutics for effective glioma management. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. 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

  3. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

    PubMed

    Allahverdian, Sima; Harada, Norihiro; Singhera, Gurpreet K; Knight, Darryl A; Dorscheid, Delbert R

    2008-02-01

    Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

  4. Remodeling of the Epithelial-Connective Tissue Interface in Oral Epithelial Dysplasia as Visualized by Noninvasive 3D Imaging.

    PubMed

    Pal, Rahul; Shilagard, Tuya; Yang, Jinping; Villarreal, Paula; Brown, Tyra; Qiu, Suimin; McCammon, Susan; Resto, Vicente; Vargas, Gracie

    2016-08-15

    Early neoplastic features in oral epithelial dysplasia are first evident at the basal epithelium positioned at the epithelial-connective tissue interface (ECTI), separating the basal epithelium from the underlying lamina propria. The ECTI undergoes significant deformation in early neoplasia due to focal epithelial expansion and proteolytic remodeling of the lamina propria, but few studies have examined these changes. In the present study, we quantitated alterations in ECTI topography in dysplasia using in vivo volumetric multiphoton autofluorescence microscopy and second harmonic generation microscopy. The label-free method allows direct noninvasive visualization of the ECTI surface without perturbing the epithelium. An image-based parameter, "ECTI contour," is described that indicates deformation of the ECTI surface. ECTI contour was higher in dysplasia than control or inflamed specimens, indicating transition from flat to a deformed surface. Cellular parameters of nuclear area, nuclear density, coefficient of variation in nuclear area in the basal epithelium and collagen density in areas adjacent to ECTI were measured. ECTI contour differentiated dysplasia from control/benign mucosa with higher sensitivity and specificity than basal nuclear density or basal nuclear area, comparable with coefficient of variation in nuclear area and collagen density. The presented method offers a unique opportunity to study ECTI in intact mucosa with simultaneous assessment of cellular and extracellular matrix features, expanding opportunities for studies of early neoplastic events near this critical interface and potentially leading to development of new approaches for detecting neoplasia in vivo Cancer Res; 76(16); 4637-47. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Microvesicles released from tumor cells disrupt epithelial cell morphology and contractility.

    PubMed

    Bordeleau, Francois; Chan, Bryan; Antonyak, Marc A; Lampi, Marsha C; Cerione, Richard A; Reinhart-King, Cynthia A

    2016-05-24

    During tumor progression, cancer cells interact and communicate with non-malignant cells within their local microenvironment. Microvesicles (MV) derived from human cancer cells play an important role in mediating this communication. Another critical aspect of cancer progression involves widespread ECM remodeling, which occur both at the primary and metastatic sites. ECM remodeling and reorganization within the tumor microenvironment is generally attributed to fibroblasts. Here, using MCF10a cells, a well-characterized breast epithelial cell line that exhibits a non-malignant epithelial phenotype, and MVs shed by aggressive MDA-MB-231 carcinoma cells, we show that non-malignant epithelial cells can participate in ECM reorganization of 3D collagen matrices following their treatment with cancer cell-derived MVs. In addition, MVs trigger several changes in epithelial cells under 3D culture conditions. Furthermore, we show that this ECM reorganization is associated with an increase in cellular traction force following MV treatment, higher acto-myosin contractility, and higher FAK activity. Overall, our findings suggest that MVs derived from tumor cells can contribute to ECM reorganization occurring within the tumor microenvironment by enhancing the contractility of non-malignant epithelial cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 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

  7. Epithelial Remodeling as Basis for Machine-Based Identification of Keratoconus

    PubMed Central

    Silverman, Ronald H.; Urs, Raksha; RoyChoudhury, Arindam; Archer, Timothy J.; Gobbe, Marine; Reinstein, Dan Z.

    2014-01-01

    Purpose. To develop and evaluate automated computerized algorithms for differentiation of normal and keratoconus corneas based solely on epithelial and stromal thickness data. Methods. Maps of the corneal epithelial and stromal thickness were generated from Artemis-1 very high-frequency ultrasound arc-scans of 130 normal and 74 keratoconic subjects diagnosed by combined topography and tomography examination. Keratoconus severity was graded based on anterior curvature, minimum corneal thickness, and refractive error. Computer analysis of maps produced 161 features for one randomly selected eye per subject. Stepwise linear discriminant analysis (LDA) and neural network (NN) analysis were then performed to develop multivariate models based on combinations of selected features to correctly classify cases. The sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) were determined for each classifier. Results. Stepwise LDA resulted in a six-variable model that provided an AUC of 100%, indicative of complete separation of keratoconic from normal corneas. Leave-one-out analysis resulted in 99.2% specificity and 94.6% sensitivity. Neural network analysis using the same six variables resulted in an AUC of 100% for the training set. Test set performance averaged over 10 trials gave a specificity of 99.5 ± 1.5% and sensitivity of 98.9 ± 1.9%. The LDA function values correlated with keratoconus severity grade. Conclusions. The results demonstrate that epithelial remodeling in keratoconus represents an independent means for differentiation of normal from advanced keratoconus corneas. PMID:24557351

  8. The Pneumocystis Ace2 transcription factor regulates cell wall-remodeling genes and organism virulence.

    PubMed

    Kottom, Theodore J; Limper, Andrew H

    2013-08-16

    Pneumocystis carinii (Pc) β-glucans are major components of the organism cell wall; yet, the regulation of Pc cell wall genesis and remodeling is not well understood. Ace2 transcription factors, which are present in many fungi, regulate glucanases and other enzymes needed for cell wall remodeling. The cloning and heterologous expression of PcAce2 in ace2Δ Saccharomyces cerevisiae demonstrated that PcAce2 can restore the defective glucanase and endochitinase gene expression of the mutant as well as regulate cell wall β-glucan biosynthetic genes. Furthermore, when a reconstructed yeast system was used, PcAce2 activated the transcription of the Pneumocystis gsc1 β-glucan synthetase, confirming the activity of a Pc transcription factor on a native Pneumocystis promoter and gene for the first time. We further observed that Pneumocystis binding to host extracellular matrix proteins and lung epithelial cells induced the phosphorylation (activation) of the PcAce2 transcription factor. Finally, we present a novel method that confirms the role of PcAce2 in modulating organism virulence using ace2Δ Candida glabrata infection in neutropenic mice. Together, these results indicate that the adherence of Pc to lung matrix proteins and epithelial cells leads to the activation of the Ace2 transcription factor, which regulates cell wall degradation and biosynthesis genes that are required for cell wall remodeling.

  9. Remodeling of cell-cell junctions in arrhythmogenic cardiomyopathy.

    PubMed

    Asimaki, Angeliki; Saffitz, Jeffrey E

    2014-02-01

    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.

  10. Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

    PubMed Central

    Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

  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. Epithelial stem cells are formed by small-particles released from particle-producing cells

    PubMed Central

    Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

    2017-01-01

    Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

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

    PubMed

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

    2013-01-01

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

  14. Patterning Bacterial Communities on Epithelial Cells

    PubMed Central

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

    2013-01-01

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

  15. Epithelial TRPV1 Signaling Accelerates Gingival Epithelial Cell Proliferation

    PubMed Central

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

    2014-01-01

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

  16. Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor.

    PubMed

    Yang, Jibing; Velikoff, Miranda; Canalis, Ernesto; Horowitz, Jeffrey C; Kim, Kevin K

    2014-04-15

    Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

  17. 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.

  18. Bone marrow-derived lung epithelial cells.

    PubMed

    Krause, Diane S

    2008-08-15

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

  19. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

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

  20. 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.

  1. Myb permits multilineage airway epithelial cell differentiation

    PubMed Central

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

    2014-01-01

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

  2. 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

  3. Purification of kidney epithelial cell growth inhibitors.

    PubMed Central

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

    1980-01-01

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

  4. Cytotoxicity and induction of inflammation by pepsin in Acid in bronchial epithelial cells.

    PubMed

    Bathoorn, Erik; Daly, Paul; Gaiser, Birgit; Sternad, Karl; Poland, Craig; Macnee, William; Drost, Ellen M

    2011-01-01

    Introduction. Gastroesophageal reflux has been associated with chronic inflammatory diseases and may be a cause of airway remodelling. Aspiration of gastric fluids may cause damage to airway epithelial cells, not only because acidity is toxic to bronchial epithelial cells, but also since it contains digestive enzymes, such as pepsin. Aim. To study whether pepsin enhances cytotoxicity and inflammation in airway epithelial cells, and whether this is pH-dependent. Methods. Human bronchial epithelial cells were exposed to increasing pepsin concentrations in varying acidic milieus, and cell proliferation and cytokine release were assessed. Results. Cell survival was decreased by pepsin exposure depending on its concentration (F = 17.4) and pH level of the medium (F = 6.5) (both P < 0.01). Pepsin-induced interleukin-8 release was greater at lower pH (F = 5.1; P < 0.01). Interleukin-6 induction by pepsin was greater at pH 1.5 compared to pH 2.5 (mean difference 434%; P = 0.03). Conclusion. Pepsin is cytotoxic to bronchial epithelial cells and induces inflammation in addition to acid alone, dependent on the level of acidity. Future studies should assess whether chronic aspiration causes airway remodelling in chronic inflammatory lung diseases.

  5. Odontogenic epithelial stem cells: hidden sources.

    PubMed

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

    2015-12-01

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

  6. Control of lens epithelial cell survival

    PubMed Central

    1993-01-01

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

  7. ATP dependent chromatin remodeling enzymes in embryonic stem cells.

    PubMed

    Saladi, Srinivas Vinod; de la Serna, Ivana L

    2010-03-01

    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.

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

    PubMed

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

    1977-12-15

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

  9. ESCRT-dependent control of membrane remodelling during cell division.

    PubMed

    Stoten, Caroline Louise; Carlton, Jeremy Graham

    2017-08-24

    The Endosomal Sorting Complex Required for Transport (ESCRT) proteins form an evolutionarily conserved membrane remodelling machinery. Identified originally for their role in cargo sorting and remodelling of endosomal membranes during yeast vacuolar sorting, an extensive body of work now implicates a sub-complex of this machinery (ESCRT-III), as a transplantable membrane fission machinery that is dispatched to various cellular locations to achieve a topologically unique membrane separation. Surprisingly, several ESCRT-III-regulated processes occur during cell division, when cells undergo a dramatic and co-ordinated remodelling of their membranes to allow the physical processes of division to occur. The ESCRT machinery functions in regeneration of the nuclear envelope during open mitosis and in the abscission phase of cytokinesis, where daughter cells are separated from each other in the last act of division. Roles for the ESCRT machinery in cell division are conserved as far back as Archaea, suggesting that the ancestral role of these proteins was as a membrane remodelling machinery that facilitated division and that was co-opted throughout evolution to perform a variety of other cell biological functions. Here, we will explore the function and regulation of the ESCRT machinery in cell division. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    PubMed

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

    2006-10-01

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

  11. Epithelial stem cells and intestinal cancer.

    PubMed

    Tan, Shawna; Barker, Nick

    2015-06-01

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

  12. Epithelial cell extrusion: Pathways and pathologies.

    PubMed

    Gudipaty, Swapna Aravind; Rosenblatt, Jody

    2016-05-19

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

  13. Physical principles of membrane remodelling during cell mechanoadaptation.

    PubMed

    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

    2015-06-15

    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.

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

    PubMed Central

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

    2010-01-01

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

  15. Propolis inhibits TGF-β1-induced epithelial-mesenchymal transition in human alveolar epithelial cells via PPARγ activation.

    PubMed

    Kao, Hui-Fang; Chang-Chien, Pei-Wen; Chang, Wen-Tsan; Yeh, Trai-Ming; Wang, Jiu-Yao

    2013-03-01

    Emerging evidence suggests that the transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to airway remodeling in severe asthma and fibrotic lung diseases. Studies have shown that extracts from propolis protect chemical-induced cardiac and liver fibrosis in animals. This study assesses the inhibitory effect of propolis on TGF-β1-induced EMT in serum-deprived A549 cells (human AECs). Experimental results show progressive cell morphological changes, decreased E-cadherin, increased N-cadherin production, intracellular F-actin rearrangement, increased reactive oxygen species (ROS) production, and increased cell motility with increasing TGF-β1 concentration. A549 cells pretreated with propolis and then treated with TGF-β1 for 24 h regained epithelial cell morphology, decreased the production of N-cadherin and ROS, and had reduced motility. Propolis prevents the effects of TGF-β1-induced Smad2 and AKT activation pathways and Snail expression. Moreover, propolis pretreatment may prevent the TGF-β1-induced down-regulation of nuclear hormone receptors and peroxisome proliferator-activated receptor gamma (PPARγ) protein in A549 cells, whose effect was blocked by adding PPARγ antagonist, GW9662. Two active components of propolis, caffeic acid phenethyl ester (CAPE) and pinocembrin (PIN), only had partial effects on TGF-β1-induced EMT in A549 cells. The results of this study suggest that natural propolis extracts may prevent TGF-β1-induced EMT in immortalized type II AECs via multiple inhibitory pathways, which may be clinically applied in the prevention and/or treatment of EMT-related fibrotic diseases as well as airway remodeling in chronic asthma. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. 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...

  17. 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...

  18. Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

    PubMed

    Glorieux, Christophe; Sandoval, Juan Marcelo; Fattaccioli, Antoine; Dejeans, Nicolas; Garbe, James C; Dieu, Marc; Verrax, Julien; Renard, Patricia; Huang, Peng; Calderon, Pedro Buc

    2016-10-01

    Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

  19. 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

  20. Uncivil engineers: Chlamydia, Salmonella and Shigella alter cytoskeleton architecture to invade epithelial cells.

    PubMed

    Dunn, Joe Dan; Valdivia, Raphael H

    2010-08-01

    The obligate intracellular bacterial pathogen Chlamydia trachomatis is a major cause of blindness and sexually transmitted diseases. Like the enteric pathogens Salmonella and Shigella, Chlamydia injects effector proteins into epithelial cells to initiate extensive remodeling of the actin cytoskeleton at the bacterial attachment site, which culminates in the engulfment of the bacterium by plasma membrane extensions. Numerous Salmonella and Shigella effectors promote this remodeling by activating Rho GTPases and tyrosine kinase signaling cascades and by directly manipulating actin dynamics. Recent studies indicate that similar host-cell alterations occur during Chlamydia invasion, but few effectors are known. The identification of additional Chlamydia effectors and the elucidation of their modes of function are critical steps towards an understanding of how this clinically important pathogen breaches epithelial surfaces and causes infection.

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

    PubMed

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

    2015-01-30

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

  2. The role of stem cells in vein graft remodelling.

    PubMed

    Xu, Q

    2007-11-01

    The vessel wall is a dynamic tissue that undergoes positive remodelling in response to altered mechanical stress. A typical example is vein graft remodelling, because veins do not develop arteriosclerosis until a vein segment is grafted on to arteries. In this process, it was observed that vascular endothelial and smooth muscle cells of vein grafts die due to suddenly elevated blood pressure. This cell death is followed by endothelial regeneration. Central to this theme is the essential role played by EPCs (endothelial progenitor cells) in regenerating the lost endothelium. The mechanisms by which EPCs attach to the vessel wall and differentiate into mature endothelial cells involve increased chemokine production and laminar shear flow stimulation on the vessel wall. It seems that neo-endothelial cells derived from EPCs lack mature cell functions and express high levels of adhesion molecules resulting in LDL (low-density lipoprotein) penetration and mononuclear cell infiltration into the sub-endothelial space. Among infiltrated mononuclear cells, there are smooth muscle progenitors that proliferate and differentiate into smooth muscle cells. Meanwhile, stem cells present in the media and adventitia may also migrate into arteriosclerotic lesions via the vasa vasorum that are abundant in the diseased vessels. However, the molecular events leading to the homing, differentiation and maturation of stem/progenitor cells still needs elucidation. The present review attempts to update the progress in stem cell research related to the pathogenesis of vein graft arteriosclerosis or remodelling, focusing on the mechanisms by which stem/progenitor cells participate in the development of lesions, and to discuss the controversial issues and the future perspectives surrounding this research area.

  3. Chromatin remodeling and bivalent histone modifications in embryonic stem cells.

    PubMed

    Harikumar, Arigela; Meshorer, Eran

    2015-12-01

    Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP-dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains.

  4. 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.

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

    PubMed Central

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

    2001-01-01

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

  6. Mammary epithelial cell: Influence of extracellular matrix composition and organization during development and tumorigenesis

    PubMed Central

    Kass, Laura; Erler, Janine T.; Dembo, Micah; Weaver, Valerie M.

    2009-01-01

    Stromal–epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal–epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation. PMID:17719831

  7. Platelet-derived microparticles and soluble factors differentially regulate human endometrial epithelial cell movement.

    PubMed

    Suginami, Koh; Sato, Yukiyasu; Horie, Akihito; Matsumoto, Hisanori; Tani, Hirohiko; Mizumoto, Yasunari; Ono, Masanori; Matsuoka, Ayumi; Kyo, Satoru; Araki, Yoshihiko; Konishi, Ikuo; Fujiwara, Hiroshi

    2017-04-01

    We previously proposed that platelets promote re-epithelialization during menstruation. As cell movement is one of the important cell behaviors in the process of tissue remodeling, we examined the effects of platelets on endometrial epithelial cell invasion. The platelets were isolated from healthy women. Using a human endometrial epithelial cell-derived immortalized cell line, EM-E6/E7/hTERT cells, we examined the effects of platelets and platelet-derived condition media with or without microparticles on the morphological and invasive properties of EM-E6/E7/hTERT cells. Platelets and microparticle-containing conditioned media inhibited Matrigel invasion by EM-E6/E7/hTERT cells along with an increase in cortical ring formation, whereas microparticle-depleted conditioned media promoted their invasion without any significant changes of cortical ring formation. These results support our previous proposal and newly suggest the dual roles of platelets: platelet-derived soluble factors that promote cell movement in the distant area, and microparticles that induce re-epithelialization by endometrial epithelial cells in the proximal area. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Phenotypic screening identifies Axl kinase as a negative regulator of an alveolar epithelial cell phenotype.

    PubMed

    Fujino, Naoya; Kubo, Hiroshi; Maciewicz, Rose A

    2017-09-01

    Loss of epithelial barrier integrity is implicated in a number of human lung diseases. However, the molecular pathways underlying this process are poorly understood. In a phenotypic screen, we identified Axl kinase as a negative regulator of epithelial phenotype and function. Furthermore, suppression of Axl activity by a small molecule kinase inhibitor or downregulation of Axl expression by small interfering RNA led to: (1) the increase in epithelial surfactant protein expression; (2) a cell morphology transition from front-rear polarity to cuboidal shape; (3) the cytoskeletal re-organization resulting in decreased cell mobility; and (4) the acquisition of epithelial junctions. Loss of Axl activity reduced activation of the Axl canonical pathway members, Akt and extracellular signal-regulated kinase-1/2 and resulted in the loss of gene expression of a unique profile of epithelial-to-mesenchymal transition transcription factors including SNAI2, HOXA5, TBX2 or TBX3. Finally, we observed that Axl was activated in hyperplasia of epithelial cells in idiopathic pulmonary fibrosis where epithelial barrier integrity was lost. These results suggest that the Axl kinase signaling pathway is associated with the loss integrity of alveolar epithelium in pathological remodeling of human lung diseases.

  9. Protons sensitize epithelial cells to mesenchymal transition.

    PubMed

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

    2012-01-01

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

  10. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

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

    2012-01-01

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

  11. Airway epithelial cells: current concepts and challenges.

    PubMed

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

    2008-09-15

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

  12. 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.

  13. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

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

    1990-01-01

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

  14. Isolation of epithelial cells with hepatobiliary phenotype.

    PubMed

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

    2008-01-01

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

  15. The human airway epithelial basal cell transcriptome.

    PubMed

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

    2011-05-04

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

  16. The Human Airway Epithelial Basal Cell Transcriptome

    PubMed Central

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

    2011-01-01

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

  17. 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

  18. 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.

  19. Reversible transdifferentiation of alveolar epithelial cells.

    PubMed

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

    1995-05-01

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

  20. Tetrahydrocurcumin Ameliorates Homocysteine Mediated Mitochondrial Remodeling in Brain Endothelial Cells.

    PubMed

    Vacek, Jonathan C; Behera, Jyotirmaya; George, Akash K; Kamat, Pradip K; Kalani, Anuradha; Tyagi, Neetu

    2017-08-18

    Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated of fission marker (DRP-1), fusion markers (Mfn2) and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) was co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15µM) ameliorated Hcy induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. 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.

  2. 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

  3. Calpain Mediates Epithelial Cell Microvillar Effacement by Enterohemorrhagic Escherichia Coli

    PubMed Central

    Lai, YuShuan; Riley, Kathleen; Cai, Andrew; Leong, John M.; Herman, Ira M.

    2011-01-01

    A member of the attaching and effacing (AE) family of pathogens, enterohemorrhagic Escherichia coli (EHEC) induces dramatic changes to the intestinal cell cytoskeleton, including effacement of microvilli. Effacement by the related pathogen enteropathogenic E. coli (EPEC) requires the activity of the Ca+2-dependent host protease, calpain, which participates in a variety of cellular processes, including cell adhesion and motility. We found that EHEC infection results in an increase in epithelial (CaCo-2a) cell calpain activity and that EHEC-induced microvillar effacement was blocked by ectopic expression of calpastatin, an endogenous calpain inhibitor, or by pretreatment of intestinal cells with a cell-penetrating version of calpastatin. In addition, ezrin, a known calpain substrate that links the plasma membrane to axial actin filaments in microvilli, was cleaved in a calpain-dependent manner during EHEC infection and lost from its normal locale within microvilli. Calpain may be a central conduit through which EHEC and other AE pathogens induce enterocyte cytoskeletal remodeling and exert their pathogenic effects. PMID:22073041

  4. Force mapping in epithelial cell migration

    PubMed Central

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

    2005-01-01

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

  5. Isolation by Size of Epithelial Tumor Cells

    PubMed Central

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

    2000-01-01

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

  6. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton.

    PubMed

    Wicki, Andreas; Lehembre, François; Wick, Nikolaus; Hantusch, Brigitte; Kerjaschki, Dontscho; Christofori, Gerhard

    2006-04-01

    The expression of podoplanin, a small mucin-like protein, is upregulated in the invasive front of a number of human carcinomas. We have investigated podoplanin function in cultured human breast cancer cells, in a mouse model of pancreatic beta cell carcinogenesis, and in human cancer biopsies. Our results indicate that podoplanin promotes tumor cell invasion in vitro and in vivo. Notably, the expression and subcellular localization of epithelial markers are unaltered, and mesenchymal markers are not induced in invasive podoplanin-expressing tumor cells. Rather, podoplanin induces collective cell migration by filopodia formation via the downregulation of the activities of small Rho family GTPases. In conclusion, podoplanin induces an alternative pathway of tumor cell invasion in the absence of epithelial-mesenchymal transition (EMT).

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

    PubMed Central

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

    1986-01-01

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

  8. Traction forces exerted by epithelial cell sheets

    NASA Astrophysics Data System (ADS)

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

    2010-05-01

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

  9. Traction forces exerted by epithelial cell sheets.

    PubMed

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

    2010-05-19

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

  10. Cells of Origin of Epithelial Ovarian Cancers

    DTIC Science & Technology

    2015-09-01

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

  11. 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

  12. Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

    PubMed

    Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

    2014-03-01

    Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

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

    PubMed Central

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

    2011-01-01

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

  15. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

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

  16. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

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

    2014-01-01

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

  17. 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

  18. TIMP-1 via TWIST1 Induces EMT Phenotypes in Human Breast Epithelial Cells

    PubMed Central

    D’Angelo, Rosemarie Chirco; Liu, Xu-Wen; Najy, Abdo J.; Jung, Young Suk; Won, Joshua; Chai, Karl X.; Fridman, Rafael; Kim, Hyeong-Reh Choi

    2014-01-01

    Tissue inhibitor of metalloproteinase-1 (TIMP1) regulates intracellular signaling networks for inhibition of apoptosis. Tetraspanin (CD63), a cell surface binding partner for TIMP-1, was previously shown to regulate integrin-mediated survival pathways in the human breast epithelial cell line MCF10A. In the current study, we show that TIMP-1 expression induces phenotypic changes in cell morphology, cell adhesion, cytoskeletal remodeling, and motility, indicative of an epithelial-mesenchymal transition (EMT). This is evidenced by loss of the epithelial cell adhesion molecule E-cadherin with an increase in the mesenchymal markers vimentin, N-cadherin, and fibronectin. Signaling through TIMP-1, but not TIMP-2, induces the expression of TWIST1, an important EMT transcription factor known to suppress E-cadherin transcription, in a CD63-dependent manner. RNAi-mediated knockdown of TWIST1 rescued E-cadherin expression in TIMP-1 overexpressing cells, demonstrating a functional significance of TWIST1 in TIMP-1 mediated EMT. Furthermore, analysis of TIMP-1 structural mutants reveals that TIMP-1 interactions with CD63 that activate cell survival signaling and EMT do not require the MMP-inhibitory domain of TIMP-1. Taken together, these data demonstrate that TIMP-1 binding to CD63 activates intracellular signal transduction pathways, resulting in EMT-like changes in breast epithelial cells, independent of its MMP-inhibitory function. PMID:24895412

  19. Control of local immunity by airway epithelial cells.

    PubMed

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

    2016-03-01

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

  20. 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

  1. ROCK1 and ROCK2 regulate epithelial polarisation and geometric cell shape.

    PubMed

    Kalaji, Ruba; Wheeler, Ann P; Erasmus, Jennifer C; Lee, Sang Y; Endres, Robert G; Cramer, Louise P; Braga, Vania M M

    2012-08-01

    Cell-cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear. Upon induction of cell-cell adhesion, we find that human keratinocytes acquire specific geometric shapes favouring hexagons, by re-modelling junction length/orientation and thus neighbour allocation. Acquisition of geometric shape correlates temporally with epithelial polarisation, as shown by an increase in lateral height. ROCK1 and ROCK2 are important regulators of myosin II contraction, but their specific role in epithelial cell shape has not been addressed. Depletion of ROCK proteins interferes with the correct proportion of hexagonal cell shapes and full elongation of lateral domain. Interestingly, ROCK proteins are not essential for maintenance of circumferential thin bundles, the main contractile epithelial F-actin pool. Instead, ROCK1 or ROCK2 regulates thin bundle contraction and positioning along the lateral domain, an important event for the stabilisation of the elongating lateral domain. Mechanistically, E-cadherin clustering specifically leads to ROCK1/ROCK2-dependent inactivation of myosin phosphatase and phosphorylation of myosin regulatory light chain. These events correlate temporally with the increase in lateral height and thin bundle compaction towards junctions. We conclude that ROCK proteins are necessary for acquisition of elongated and geometric cell shape, two key events for epithelial differentiation. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  2. 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.

  3. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

    PubMed Central

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

    2013-01-01

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

  4. Chromatin remodeling by the small RNA machinery in mammalian cells.

    PubMed

    Li, Long-Cheng

    2014-01-01

    Chromatin states, quite different from changes in DNA sequence, can impact fundamental cellular processes such as determination of cell identity and development of disease. However, how chromatin states are established and regulated remain to be fully elucidated. In several lower eukaryotes, the small RNA machinery comprised of small RNA and its partners, the Argonaute proteins, is known to play important roles in the establishment of heterochromatin and silencing of repetitive sequences. In mammalian cells, however, the nuclear function of the small RNA machinery is largely unknown. Emerging evidence suggests that components of the small RNA pathway interact with chromatin to regulate nuclear events, including gene transcription and alternative splicing. In addition, these endogenous mechanisms are being exploited to target specific genomic loci for manipulation of gene expression and splicing events. In this review, I summarize current understanding of chromatin remodeling by small RNAs in mammalian cells and highlight recent efforts to map genome-wide interactions between RNAi-related factors and chromatin.

  5. Complex epithelial remodeling underlie the fusion event in early fetal development of the human penile urethra.

    PubMed

    Shen, Joel; Overland, Maya; Sinclair, Adriane; Cao, Mei; Yue, Xuan; Cunha, Gerald; Baskin, Laurence

    We recently described a two-step process of urethral plate canalization and urethral fold fusion to form the human penile urethra. Canalization ("opening zipper") opens the solid urethral plate into a groove, and fusion ("closing zipper") closes the urethral groove to form the penile urethra. We hypothesize that failure of canalization and/or fusion during human urethral formation can lead to hypospadias. Herein, we use scanning electron microscopy (SEM) and analysis of transverse serial sections to better characterize development of the human fetal penile urethra as contrasted to the development of the human fetal clitoris. Eighteen 7-13 week human fetal external genitalia specimens were analyzed by SEM, and fifteen additional human fetal specimens were sectioned for histologic analysis. SEM images demonstrate canalization of the urethral/vestibular plate in the developing male and female external genitalia, respectively, followed by proximal to distal fusion of the urethral folds in males only. The fusion process during penile development occurs sequentially in multiple layers and through the interlacing of epidermal "cords". Complex epithelial organization is also noted at the site of active canalization. The demarcation between the epidermis of the shaft and the glans becomes distinct during development, and the epithelial tag at the distal tip of the penile and clitoral glans regresses as development progresses. In summary, SEM analysis of human fetal specimens supports the two-zipper hypothesis of formation of the penile urethra. The opening zipper progresses from proximal to distal along the shaft of the penis and clitoris into the glans in identical fashion in both sexes. The closing zipper mechanism is active only in males and is not a single process but rather a series of layered fusion events, uniquely different from the simple fusion of two epithelial surfaces as occurs in formation of the palate and neural tube.

  6. Complex epithelial remodeling underlie the fusion event in early fetal development of the human penile urethra

    PubMed Central

    Sinclair, Adriane; Cao, Mei; Yue, Xuan; Cunha, Gerald; Baskin, Laurence

    2016-01-01

    We recently described a two-step process of urethral plate canalization and urethral fold fusion to form the human penile urethra. Canalization (“opening zipper”) opens the solid urethral plate into a groove, and fusion (“closing zipper”) closes the urethral groove to form the penile urethra. We hypothesize that failure of canalization and/or fusion during human urethral formation can lead to hypospadias. Herein, we use scanning electron microscopy (SEM) and analysis of transverse serial sections to better characterize development of the human fetal penile urethra as contrasted to the development of the human fetal clitoris. Eighteen 7-13 week human fetal external genitalia specimens were analyzed by SEM, and fifteen additional human fetal specimens were sectioned for histologic analysis. SEM images demonstrate canalization of the urethral/vestibular plate in the developing male and female external genitalia, respectively, followed by proximal to distal fusion of the urethral folds in males only. The fusion process during penile development occurs sequentially in multiple layers and through the interlacing of epidermal “cords”. Complex epithelial organization is also noted at the site of active canalization. The demarcation between the epidermis of the shaft and the glans becomes distinct during development, and the epithelial tag at the distal tip of the penile and clitoral glans regresses as development progresses. In summary, SEM analysis of human fetal specimens supports the two-zipper hypothesis of formation of the penile urethra. The opening zipper progresses from proximal to distal along the shaft of the penis and clitoris into the glans in identical fashion in both sexes. The closing zipper mechanism is active only in males and is not a single process but rather a series of layered fusion events, uniquely different from the simple fusion of two epithelial surfaces as occurs in formation of the palate and neural tube. PMID:27397682

  7. 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.

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

    PubMed

    Wilson, Tyler J; Bergstralh, Dan T

    2017-06-01

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

  9. Phenotypic plasticity in normal breast derived epithelial cells

    PubMed Central

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  11. 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

  12. 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

  13. Cell death, remodeling, and repair in chronic obstructive pulmonary disease?

    PubMed

    Henson, Peter M; Vandivier, R William; Douglas, Ivor S

    2006-11-01

    Apoptotic cells can be detected in the parenchyma and airways of patients with chronic obstructive pulmonary disease (COPD) in greater numbers than seen in normal lungs or those from smokers without COPD. Implications include more apoptosis and/or decreased clearance of apoptotic cells. Both epithelial and endothelial cells become apoptotic. What role does the apoptosis play in the emphysema or small airway alterations seen in COPD? In simple terms, loss of cells by apoptosis would be expected to accompany, or perhaps initiate, the overall tissue destruction normally believed responsible. Indeed, direct induction of apoptosis in pulmonary endothelial or epithelial cells in rodents is accompanied by emphysematous changes. On the other hand, apoptotic cells are normally removed from tissues rapidly with minimal tissue response, to be followed by cell replacement to maintain homeostasis. The presence of detectable apoptotic cells, therefore, may imply defects in these clearance mechanisms, and, in keeping with this hypothesis, there is increasing evidence for such defects in patients with COPD. Mice with abnormalities in apoptotic cell removal also tend to develop spontaneous "emphysema." A reconciling hypothesis is that recognition of apoptotic cells not only leads to removal but also, normally, to signals for cell replacement. If this latter response is lacking in COPD-susceptible smokers, defects in normal alveolar or small airway repair could significantly contribute to the structural disruption. The concept puts emphasis on defective repair as well as initial injury (i.e., persistent alteration of dynamic tissue homeostasis, as a key contributor to COPD), with, it is hoped, additional approaches for mitigation.

  14. Epigenetics in Intestinal Epithelial Cell Renewal

    PubMed Central

    Roostaee, Alireza; Benoit, Yannick D.; Boudjadi, Salah

    2016-01-01

    A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. J. Cell. Physiol. 231: 2361–2367, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27061836

  15. Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis

    PubMed Central

    Gifford, Gail B; Demitrack, Elise S; Keeley, Theresa M; Tam, Andrew; La Cunza, Nilsa; Dedhia, Priya H; Spence, Jason R; Simeone, Diane M; Saotome, Ichiko; Louvi, Angeliki; Siebel, Christian W; Samuelson, Linda C

    2016-01-01

    Objective We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue. Design Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors. Results Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth. Conclusions Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach. PMID:26933171

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2013-12-01

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

  18. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

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

  19. 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.

  20. Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells

    PubMed Central

    Wang, Yuan Yuan; Attané, Camille; Milhas, Delphine; Dirat, Béatrice; Dauvillier, Stéphanie; Guerard, Adrien; Gilhodes, Julia; Lazar, Ikrame; Alet, Nathalie; Laurent, Victor; Le Gonidec, Sophie; Hervé, Caroline; Bost, Frédéric; Ren, Guo Sheng; Bono, Françoise; Escourrou, Ghislaine; Prentki, Marc; Nieto, Laurence; Valet, Philippe

    2017-01-01

    In breast cancer, a key feature of peritumoral adipocytes is their loss of lipid content observed both in vitro and in human tumors. The free fatty acids (FFAs), released by adipocytes after lipolysis induced by tumor secretions, are transferred and stored in tumor cells as triglycerides in lipid droplets. In tumor cell lines, we demonstrate that FFAs can be released over time from lipid droplets through an adipose triglyceride lipase–dependent (ATGL-dependent) lipolytic pathway. In vivo, ATGL is expressed in human tumors where its expression correlates with tumor aggressiveness and is upregulated by contact with adipocytes. The released FFAs are then used for fatty acid β-oxidation (FAO), an active process in cancer but not normal breast epithelial cells, and regulated by coculture with adipocytes. However, in cocultivated cells, FAO is uncoupled from ATP production, leading to AMPK/acetyl-CoA carboxylase activation, a circle that maintains this state of metabolic remodeling. The increased invasive capacities of tumor cells induced by coculture are completely abrogated by inhibition of the coupled ATGL-dependent lipolysis/FAO pathways. These results show a complex metabolic symbiosis between tumor-surrounding adipocytes and cancer cells that stimulate their invasiveness, highlighting ATGL as a potential therapeutic target to impede breast cancer progression. PMID:28239646

  1. 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

  2. Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation

    PubMed Central

    Ojkic, Nikola; López-Garrido, Javier; Pogliano, Kit; Endres, Robert G

    2016-01-01

    When starved, the Gram-positive bacterium Bacillus subtilis forms durable spores for survival. Sporulation initiates with an asymmetric cell division, creating a large mother cell and a small forespore. Subsequently, the mother cell membrane engulfs the forespore in a phagocytosis-like process. However, the force generation mechanism for forward membrane movement remains unknown. Here, we show that membrane migration is driven by cell wall remodeling at the leading edge of the engulfing membrane, with peptidoglycan synthesis and degradation mediated by penicillin binding proteins in the forespore and a cell wall degradation protein complex in the mother cell. We propose a simple model for engulfment in which the junction between the septum and the lateral cell wall moves around the forespore by a mechanism resembling the ‘template model’. Hence, we establish a biophysical mechanism for the creation of a force for engulfment based on the coordination between cell wall synthesis and degradation. DOI: http://dx.doi.org/10.7554/eLife.18657.001 PMID:27852437

  3. PREPARATION OF EPITHELIAL AND MESENCHYMAL STEM CELLS FROM MURINE MAMMARY GLAND

    PubMed Central

    Guest, I.; Ilic, Z.; Ma, J.

    2011-01-01

    The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for this remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44 and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumour development. PMID:22058055

  4. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

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

    2006-01-01

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

  5. Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

    PubMed

    Andarawewa, Kumari L; Erickson, Anna C; Chou, William S; Costes, Sylvain V; Gascard, Philippe; Mott, Joni D; Bissell, Mina J; Barcellos-Hoff, Mary Helen

    2007-09-15

    Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  6. [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).

  7. Cell death and tissue remodeling in planarian regeneration.

    PubMed

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

    2010-02-01

    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 initial 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.

  8. Cation Type Specific Cell Remodeling Regulates Attachment Strength

    PubMed Central

    Fuhrmann, Alexander; Li, Julie; Chien, Shu; Engler, Adam J.

    2014-01-01

    Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α5-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions. PMID:25014042

  9. 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

  10. Role of mesenchymal cell death in lung remodeling after injury.

    PubMed Central

    Polunovsky, V A; Chen, B; Henke, C; Snover, D; Wendt, C; Ingbar, D H; Bitterman, P B

    1993-01-01

    Repair after acute lung injury requires elimination of granulation tissue from the alveolar airspace. We hypothesized that during lung repair, signals capable of inducing the death of the two principal cellular elements of granulation tissue, fibroblasts and endothelial cells, would be present at the air-lung interface. Bronchoalveolar lavage fluid obtained from patients during lung repair induced both fibroblast and endothelial cell death, while fluid obtained at the time of injury or from patient controls did not. The mode of cell death for endothelial cells was apoptosis. Fibroblast death, while morphologically distinct from necrosis, also differed from typical apoptosis. Only proliferating cells were susceptible to the bioactivities in lavage fluid, which were trypsin sensitive and lipid insoluble. Histological examination of lung tissue from patients after lung injury revealed evidence of apoptotic cells within airspace granulation tissue. Our results suggest that cell death induced by peptide(s) present at the air-lung interface may participate in the remodeling process that accompanies tissue repair after injury. Images PMID:8326006

  11. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

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

    2013-03-01

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

  12. 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

  13. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

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

  14. 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

  15. F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells

    PubMed Central

    Feygin, Alex; Ivanov, Andrei I.

    2015-01-01

    Tight junctions (TJ) and adherens junctions (AJ) are key morphological features of differentiated epithelial cells that regulate the integrity and permeability of tissue barriers. Structure and remodeling of epithelial junctions depends on their association with the underlying actomyosin cytoskeleton. Anillin is a unique scaffolding protein interacting with different cytoskeletal components, including actin filaments and myosin motors. Its role in the regulation of mammalian epithelial junctions remains unexplored. Downregulation of anillin expression in human prostate, colonic, and lung epithelial cells triggered AJ and TJ disassembly without altering the expression of junctional proteins. This junctional disassembly was accompanied by dramatic disorganization of the perijunctional actomyosin belt; while the general architecture of the actin cytoskeleton, and activation status of non-muscle myosin II, remained unchanged. Furthermore, loss of anillin disrupted the adducin-spectrin membrane skeleton at the areas of cell-cell contact, selectively decreased γ-adducin expression, and induced cytoplasmic aggregation of αII-spectrin. Anillin knockdown activated c-Jun N-terminal kinase (JNK), and JNK inhibition restored AJ and TJ integrity and cytoskeletal organization in anillin-depleted cells. These findings suggest a novel role for anillin in regulating intercellular adhesion in model human epithelia by mechanisms involving the suppression of JNK activity and controlling the assembly of the perijunctional cytoskeleton. PMID:25809162

  16. Role of β-Pix in corneal epithelial cell migration on fibronectin.

    PubMed

    Kimura, Kazuhiro; Teranishi, Shinichiro; Orita, Tomoko; Zhou, Hongyan; Nishida, Teruo

    2011-05-16

    Corneal epithelial migration during wound healing is important for maintenance of corneal transparency, and fibronectin plays a key role in regulation of the adhesion and migration of corneal epithelial cells. The role of β-Pix in intracellular signaling that underlies the stimulatory effects of fibronectin on the adhesion and migration of corneal epithelial cells was examined. Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured on fibronectin or on bovine serum albumin as a control. The localization and tyrosine phosphorylation of β-Pix were examined by immunofluorescence and immunoprecipitation analyses, respectively. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to phosphotyrosine, respectively. The effects of depletion of β-Pix on HCE cell adhesion and migration on fibronectin were investigated by cell transfection with a small interfering RNA specific for β-Pix mRNA. Fibronectin induced the tyrosine phosphorylation of β-Pix as well as its apparent accumulation at focal adhesions in HCE cells. Depletion of β-Pix inhibited the effects of fibronectin on remodeling of the actin cytoskeleton and the formation of focal adhesions. It also inhibited the migration of HCE cells on fibronectin in an in vitro model of wound healing, but it did not affect cell adhesion to fibronectin. β-Pix contributes to the regulation of the formation of focal adhesions as well as that of cell migration by fibronectin in HCE cells. This protein therefore likely plays an important role in signal transduction underlying corneal epithelial wound healing.

  17. 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.

  18. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling.

    PubMed

    Salmon, M; Walsh, D A; Koto, H; Barnes, P J; Chung, K F

    1999-09-01

    Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.

  19. 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.

  20. Epithelial Cell Innate Response to Candida albicans

    PubMed Central

    Naglik, J.R.; Moyes, D.

    2011-01-01

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

  1. 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

    2016-02-04

    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.

  2. 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

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

    PubMed

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

    2009-11-02

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

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

    PubMed Central

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

    2014-01-01

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

  5. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

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

  6. Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

    PubMed Central

    Ventoso, Iván; Kochetov, Alex; Montaner, David; Dopazo, Joaquín; Santoyo, Javier

    2012-01-01

    In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed. PMID

  7. 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

  8. Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

    PubMed Central

    Shiraishi, Takumi; Verdone, James E.; Huang, Jessie; Kahlert, Ulf D.; Hernandez, James R.; Torga, Gonzalo; Zarif, Jelani C.; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H.; Mooney, Steven M.; An, Steven S.; Pienta, Kenneth J.

    2015-01-01

    The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease. PMID:25426557

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

    PubMed Central

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

    2017-01-01

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

  10. 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

  11. 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

  12. The butterfly effect in cancer: a single base mutation can remodel the cell.

    PubMed

    Hart, Jonathan R; Zhang, Yaoyang; Liao, Lujian; Ueno, Lynn; Du, Lisa; Jonkers, Marloes; Yates, John R; Vogt, Peter K

    2015-01-27

    We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.

  13. 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

  14. 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

  15. 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

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

    PubMed

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

    2017-03-02

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

  17. Nicotine-induced epithelial-mesenchymal transition via Wnt/β-catenin signaling in human airway epithelial cells.

    PubMed

    Zou, Weifeng; Zou, Yimin; Zhao, Zhuxiang; Li, Bing; Ran, Pixin

    2013-02-15

    Epithelial-mesenchymal transition (EMT) has been proposed to be a mechanism in airway remodeling, which is a characteristic of chronic obstructive pulmonary disease (COPD). Studies have shown that cigarette smoke and nicotine are factors that induce Wnt/β-catenin activation, which is a pathway that has also been implicated in EMT. The main aim of this study was to test whether human bronchial epithelial cells are able to undergo EMT in vitro following nicotine stimulation via the Wnt3a/β-catenin signaling pathway. We show that nicotine activates the Wnt3a signal pathway, which leads to the translocation of β-catenin into the nucleus and activation of β-catenin/Tcf-dependent transcription in the human bronchial epithelial cell (HBEC) line. This accumulation was accompanied by an increase in smooth muscle actin, vimentin, matrix metalloproteinases-9, and type I collagen expression as well as downregulation of E-cadherin, which are typical characteristics of EMT. We also noted that the release of TGF-β(1) from these cells was stimulated by nicotine. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented these effects, implying that β-catenin activation in these responses is Wnt3a dependent. Furthermore, specific knockdown of TGF-β(1) with TGF-β(1) siRNA partially prevented nicotine-induced EMT, suggesting that TGF-β(1) has a role in nicotine-mediated EMT in HBECs. These results suggest that HBECs are able to undergo EMT in vitro upon nicotine stimulation via the Wnt3a/β-catenin signaling pathway.

  18. 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.

  19. Polarity in Mammalian Epithelial Morphogenesis

    PubMed Central

    Roignot, Julie; Peng, Xiao; Mostov, Keith

    2013-01-01

    Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires the intervention of several fundamental cell processes, whose integration in space and time is only starting to be elucidated. To understand what governs the building of epithelial tissues during development, it is essential to consider the polarization process in the context of the whole tissue. To this end, the development of three-dimensional organotypic cell culture models has brought new insights into the mechanisms underlying the establishment and maintenance of higher-order epithelial tissue architecture, and in the dynamic remodeling of cell polarity that often occurs during development of epithelial organs. Here we discuss some important aspects of mammalian epithelial morphogenesis, from the establishment of cell polarity to epithelial tissue generation. PMID:23378592

  20. 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

  1. Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds.

    PubMed

    Cerqueira, M T; Pirraco, R P; Martins, A R; Santos, T C; Reis, R L; Marques, A P

    2014-07-01

    Skin regeneration remains a challenge, requiring a well-orchestrated interplay of cell-cell and cell-matrix signalling. Cell sheet (CS) engineering, which has the major advantage of allowing the retrieval of the intact cell layers along with their naturally organized extracellular matrix (ECM), has been poorly explored for the purpose of creating skin substitutes and skin regeneration. This work proposes the use of CS technology to engineer cellular constructs based on human keratinocytes (hKC), key players in wound re-epithelialization, dermal fibroblasts (hDFb), responsible for ECM remodelling, and dermal microvascular endothelial cells (hDMEC), part of the dermal vascular network and modulators of angiogenesis. Homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed simultaneously to target wound re-vascularization and re-epithelialization. After implantation of the constructs in murine full-thickness wounds, human cells were engrafted into the host wound bed and were present in the neotissue formed up to 14 days post-implantation. Different outcomes were obtained by varying the composition and organization of the 3-D constructs. Both hKC and hDMEC significantly contributed to re-epithelialization by promoting rapid wound closure and early epithelial coverage. Moreover, a significant increase in the density of vessels at day 7 and the incorporation of hDMEC in the neoformed vasculature confirmed its role over neotissue vacularization. As a whole, the obtained results confirmed that the proposed 3-D CS-based constructs provided the necessary cell machinery, when in a specific microenvironment, guiding both re-vascularization and re-epithelialization. Although dependent on the nature of the constructs, the results obtained sustain the hypothesis that different CS-based constructs lead to improved skin healing.

  2. Corneal epithelial stem cells: deficiency and regulation.

    PubMed

    Secker, Genevieve A; Daniels, Julie T

    2008-09-01

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

  3. 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

  4. 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

  5. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

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

    2016-04-01

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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Mast cell desensitization inhibits calcium flux and aberrantly remodels actin

    PubMed Central

    Ang, W.X. Gladys; Church, Alison M.; Kulis, Mike; Choi, Hae Woong; Burks, A. Wesley

    2016-01-01

    Rush desensitization (DS) is a widely used and effective clinical strategy for the rapid inhibition of IgE-mediated anaphylactic responses. However, the cellular targets and underlying mechanisms behind this process remain unclear. Recent studies have implicated mast cells (MCs) as the primary target cells for DS. Here, we developed a murine model of passive anaphylaxis with demonstrated MC involvement and an in vitro assay to evaluate the effect of DS on MCs. In contrast with previous reports, we determined that functional IgE remains on the cell surface of desensitized MCs following DS. Despite notable reductions in MC degranulation following DS, the high-affinity IgE receptor FcεRI was still capable of transducing signals in desensitized MCs. Additionally, we found that displacement of the actin cytoskeleton and its continued association with FcεRI impede the capacity of desensitized MCs to evoke the calcium response that is essential for MC degranulation. Together, these findings suggest that reduced degranulation responses in desensitized MCs arise from aberrant actin remodeling, providing insights that may lead to improvement of DS treatments for anaphylactic responses. PMID:27669462

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

    PubMed

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

    2008-04-01

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

  13. Osteopontin traffic in hypoxic renal epithelial cells.

    PubMed

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

    2003-01-01

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

  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. 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

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

    PubMed

    Huang, Xun; Yan, Xiaolong; Zhang, Zhipei; Li, Xiaofei

    2015-06-01

    The specific role and mechanism of epithelium in the progression of obliterative airway disease (OAD) after tracheal allotransplantation remain poorly understood. In this study, we used rat heterotopic tracheal transplantation to investigate the mechanism of epithelial cell seeding during the process of OAD. Prospective, basic science. Research laboratory. In total, 120 Sprague Dawley (SD) rats and 90 Wistar rats were used. Tracheas from SD rats were implanted into SD rats (syngeneic, n = 30) or Wistar rats (allogeneic, n = 30), and SD rat tracheas (seeded with Wistar rat-derived epithelial cells 6 days after transplantation) were transplanted into Wistar rats (seeded allogeneic, n = 30). Grafts were harvested at 7, 14, or 30 days after transplantation for histologic, quantitative reverse transcriptional polymerase chain reaction or Western blot analyses. Syngrafts retained normal histologic structures, while the corresponding allografts demonstrated less ciliated epithelia and more lumenal occlusion. Seeding of epithelial cells ameliorated the histologic changes, reduced the expression of epithelial to mesenchymal transition (EMT)-related transcriptional factors and mesenchymal markers, and dampened the expression of transforming growth factor β1 (TGF-β1) and phosphorylation of smad3. Seeding of recipient epithelial cells inhibits the progression of OAD by attenuating EMT via TGF-β-Smad signaling in rat heterotopic tracheal allografts. Clinically, the injection of recipient-originated epithelial cells might provide new insights into the treatment for OAD after tracheal allotransplantation. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

  17. Cytomatrix synthesis in MDCK epithelial cells

    SciTech Connect

    Mitchell, J.J.; Low, R.B.; Woodcock-Mitchell, J.L. )

    1990-06-01

    Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with (14C)leucine over several days and then pulse-labeled for 4 hours with (3H)leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form.

  18. Cervical Remodeling/Ripening at Term and Preterm Delivery: The Same Mechanism Initiated by Different Mediators and Different Effector Cells

    PubMed Central

    Gonzalez, Juan M.; Dong, Zhong; Romero, Roberto; Girardi, Guillermina

    2011-01-01

    Background Premature cervical remodeling/ripening is believed to contribute to preterm delivery (PTD), the leading cause of perinatal morbidity and mortality. Despite considerable research, the causes of term and PTD remain unclear, and there is no effective treatment for PTD. We previously demonstrated that complement activation plays a causative role in cervical remodeling that leads to PTD in mice. Methodology/Principal Findings Here we found that complement activation is not required for the physiological process that leads to term delivery in mice. Neither increased C3 cervical deposition nor increased C3a and C5a serum levels were observed at term. In addition, macrophages infiltration was found in PTD in contrast to term delivery were no leukocytes were found. Despite the different role of complement and different cellular effector cells, PTD and term delivery share a common dowsntream pathway characterized by increased metalloproteinases (MMPs) release and increased collagen degradation. However, different sources of MMPs were identified. Macrophages are the source of MMPs in PTD while cervical fibroblasts and columnar epithelial cells synthesize MMPs at term delivery. A dramatic diminution in serum progesterone levels precedes parturition at term but not in PTD, suggesting that progesterone withdrawal initiates cervical remodeling at term. On the other hand, MMPs release in PTD is triggered by C5a. Conclusion and Significance In conclusion, preterm and term cervical remodeling occur through the same mechanism but they are initiated by different mediators and effector cells. That complement activation is required for PTD but not for the physiological process that leads to term delivery, suggests that complement is a potential specific biomarker and selective target to prevent PTD and thus avert neonatal mortality and morbidity. PMID:22073213

  19. 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.

  20. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  1. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

    PubMed

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-07-15

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

  2. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

    PubMed Central

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-01-01

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite. PMID:25221599

  3. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  4. Cell death at the intestinal epithelial front line.

    PubMed

    Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

    2016-07-01

    The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

  5. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    PubMed Central

    Rodrigues-Diez, Raquel; Rodrigues-Diez, Raúl R.; Lavoz, Carolina; Carvajal, Gisselle; Droguett, Alejandra; Garcia-Redondo, Ana B.; Rodriguez, Isabel; Ortiz, Alberto; Egido, Jesús; Mezzano, Sergio; Ruiz-Ortega, Marta

    2014-01-01

    Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β. PMID:24949470

  6. Epithelial-to-mesenchymal transition in penile squamous cell carcinoma.

    PubMed

    Masferrer, Emili; Ferrándiz-Pulido, Carla; Masferrer-Niubò, Magalí; Rodríguez-Rodríguez, Alfredo; Gil, Inmaculada; Pont, Antoni; Servitje, Octavi; García de Herreros, Antonio; Lloveras, Belen; García-Patos, Vicenç; Pujol, Ramon M; Toll, Agustí; Hernández-Muñoz, Inmaculada

    2015-02-01

    Epithelial-to-mesenchymal transition is a phenomenon in epithelial tumors that involves loss of intercellular adhesion, mesenchymal phenotype acquisition and enhanced migratory potential. While the epithelial-to-mesenchymal transition process has been extensively linked to metastatic progression of squamous cell carcinoma, studies of the role of epithelial-to-mesenchymal transition in squamous cell carcinoma containing high risk human papillomaviruses are scarce. Moreover, to our knowledge epithelial-to-mesenchymal transition involvement in human penile squamous cell carcinoma, which can arise through transforming HPV infections or independently of HPV, has not been investigated. We evaluated the presence of epithelial-to-mesenchymal transition markers and their relationship to HPV in penile squamous cell carcinoma. We assessed the expression of E-cadherin, vimentin and the epithelial-to-mesenchymal transition related transcription factors Twist, Zeb1 and Snail by immunohistochemical staining in 64 penile squamous cell carcinoma cases. HPV was detected by polymerase chain reaction amplification. Simultaneous loss of membranous E-cadherin expression and vimentin over expression were noted in 43.5% of penile squamous cell carcinoma cases. HPV was significantly associated with loss of membranous E-cadherin but not with epithelial-to-mesenchymal transition. Recurrence and mortality rates were significantly higher in cases showing epithelial-to-mesenchymal transition. Our findings indicate that in penile squamous cell carcinoma epithelial-to-mesenchymal transition is associated with poor prognosis but not with the presence of HPV. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  7. 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

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

    PubMed

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

    2015-02-13

    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.

  9. EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity.

    PubMed

    Tapia, Rocio; Kralicek, Sarah E; Hecht, Gail A

    2017-06-15

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1-integrin and Na(+) /K(+) ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na(+) /K(+) ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na(+) /K(+) ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical-basal polarity in an EspF-dependent manner, which would contribute to EPEC-associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes. © 2017 John Wiley & Sons Ltd.

  10. 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

  11. 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.

  12. Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

    PubMed Central

    Knox, Alan J.

    2015-01-01

    Human airway smooth muscle cells (HASMC) contribute to asthma pathophysiology through an increased smooth muscle mass and elevated cytokine/chemokine output. Little is known about how HASMC and the airway epithelium interact to regulate chronic airway inflammation and remodeling. Amphiregulin is a member of the family of epidermal growth factor receptor (EGFR) agonists with cell growth and proinflammatory roles and increased expression in the lungs of asthma patients. Here we show that bradykinin (BK) stimulation of HASMC increases amphiregulin secretion in a mechanism dependent on BK-induced COX-2 expression, increased PGE2 output, and the stimulation of HASMC EP2 and EP4 receptors. Conditioned medium from BK treated HASMC induced CXCL8, VEGF, and COX-2 mRNA and protein accumulation in airway epithelial cells, which were blocked by anti-amphiregulin antibodies and amphiregulin siRNA, suggesting a paracrine effect of HASMC-derived amphiregulin on airway epithelial cells. Consistent with this, recombinant amphiregulin induced CXCL8, VEGF, and COX-2 in airway epithelial cells. Finally, we found that conditioned media from amphiregulin-stimulated airway epithelial cells induced amphiregulin expression in HASMC and that this was dependent on airway epithelial cell COX-2 activity. Our study provides evidence of a dynamic axis of interaction between HASMC and epithelial cells that amplifies CXCL8, VEGF, COX-2, and amphiregulin production. PMID:26047642

  13. Clinical implications of epithelial cell plasticity in cancer progression.

    PubMed

    Aparicio, Luis A; Blanco, Moisés; Castosa, Raquel; Concha, Ángel; Valladares, Manuel; Calvo, Lourdes; Figueroa, Angélica

    2015-09-28

    In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients.

  14. Human Growth Hormone Promotes Corneal Epithelial Cell Migration in Vitro

    PubMed Central

    Ding, Juan; Wirostko, Barbara; Sullivan, David A

    2015-01-01

    Purpose Corneal wound healing is a highly regulated process that requires the proliferation and migration of epithelial cells and interactions between epithelial cells and stromal fibroblasts. Compounds that can be applied topically to the ocular surface and that have the capability of activating corneal epithelial cells to proliferate and/or migrate would be useful to promote corneal wound healing. We hypothesize that human growth hormone (HGH) will activate Signal Transducer and Activators of Transcription-5 (STAT5) signaling and promote corneal wound healing by enhancing corneal epithelial cell and fibroblast proliferation and/or migration in vitro. The purpose of this study is to test these hypotheses. Methods We studied cell signaling, proliferation and migration using an immortalized human corneal epithelial cell line and primary human corneal fibroblasts in vitro. We also examined whether insulin-like growth factor-1 (IGF-1), a hormone known to mediate many of HGH’s growth promoting actions, may play a role in this effect. Results We show that HGH activates STAT5 signaling and promotes corneal epithelial cell migration in vitro. The migratory effect requires an intact communication between corneal epithelia and fibroblasts, and is not mediated by IGF-1. Conclusion HGH may represent a topical therapeutic to promote corneal epithelial wound healing. This warrants further investigation. PMID:25782399

  15. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model.

    PubMed

    Carey, Shawn P; Starchenko, Alina; McGregor, Alexandra L; Reinhart-King, Cynthia A

    2013-06-01

    Solid tumors consist of genetically and phenotypically diverse subpopulations of cancer cells with unique capacities for growth, differentiation, and invasion. While the molecular and microenvironmental bases for heterogeneity are increasingly appreciated, the outcomes of such intratumor heterogeneity, particularly in the context of tumor invasion and metastasis, remain poorly understood. To study heterotypic cell-cell interactions and elucidate the biological consequences of intratumor heterogeneity, we developed a tissue-engineered multicellular spheroid (MCS) co-culture model that recapitulates the cellular diversity and fully three-dimensional cell-cell and cell-matrix interactions that characterize human carcinomas. We found that "invasion-competent" malignant cells induced the collective invasion of otherwise "invasion-incompetent" epithelial cells, and that these two cell types consistently exhibited distinct leader and follower roles during invasion. Analysis of extracellular matrix (ECM) microarchitecture revealed that malignant cell invasion was accompanied by extensive ECM remodeling including matrix alignment and proteolytic track-making. Inhibition of cell contractility- and proteolysis-mediated matrix reorganization prevented leader-follower behavior and malignant cell-induced epithelial cell invasion. These results indicate that heterogeneous subpopulations within a tumor may possess specialized roles during tumor progression and suggest that complex interactions among the various subpopulations of cancer cells within a tumor may regulate critical aspects of tumor biology and affect clinical outcome.

  16. Differential HIF-1α and HIF-2α Expression in Mammary Epithelial Cells during Fat Pad Invasion, Lactation, and Involution.

    PubMed

    Påhlman, Sven; Lund, Leif R; Jögi, Annika

    2015-01-01

    The development and functional cycle of the mammary gland involves a number of processes that are caricatured by breast cancer cells during invasion and metastasis. Expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 has been associated with metastatic, poor prognosis, and high-grade breast cancers. Since hypoxia affects normal epithelial differentiation, we hypothesise that HIFs are important for normal breast epithelial development and regeneration as well as cancer initiation and progression. Here, we investigated the expression of the oxygen-sensitive HIF-alpha subunits during mouse mammary gland development, lactation, and involution. In breast epithelial cells, HIF-1α was expressed during early development, prior to cell polarisation. In contrast, expression of HIF-2α occurred later and was restricted to a subpopulation of luminal epithelial cells in the lactating gland. Mammary gland involution is a developmental stage that involves extensive tissue remodelling with cell death but survival of tissue stem/progenitor cells. At this stage, HIF-2α, but little HIF-1α, was expressed in CK14-positive epithelial cells. The temporal but differential expression of the HIF-alpha subunits during the mammary gland life cycle indicates that their expression is controlled by additional factors to hypoxia. Further functional studies of the roles of these proteins in the mammary gland and breast cancer are warranted.

  17. Differential HIF-1α and HIF-2α Expression in Mammary Epithelial Cells during Fat Pad Invasion, Lactation, and Involution

    PubMed Central

    Påhlman, Sven; Lund, Leif R.; Jögi, Annika

    2015-01-01

    The development and functional cycle of the mammary gland involves a number of processes that are caricatured by breast cancer cells during invasion and metastasis. Expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 has been associated with metastatic, poor prognosis, and high-grade breast cancers. Since hypoxia affects normal epithelial differentiation, we hypothesise that HIFs are important for normal breast epithelial development and regeneration as well as cancer initiation and progression. Here, we investigated the expression of the oxygen-sensitive HIF-alpha subunits during mouse mammary gland development, lactation, and involution. In breast epithelial cells, HIF-1α was expressed during early development, prior to cell polarisation. In contrast, expression of HIF-2α occurred later and was restricted to a subpopulation of luminal epithelial cells in the lactating gland. Mammary gland involution is a developmental stage that involves extensive tissue remodelling with cell death but survival of tissue stem/progenitor cells. At this stage, HIF-2α, but little HIF-1α, was expressed in CK14-positive epithelial cells. The temporal but differential expression of the HIF-alpha subunits during the mammary gland life cycle indicates that their expression is controlled by additional factors to hypoxia. Further functional studies of the roles of these proteins in the mammary gland and breast cancer are warranted. PMID:25955753

  18. 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

  19. 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.

  20. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

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

    PubMed

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

    2013-06-27

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

  2. Cultured epithelial cells response to phototherapy with low intensity laser.

    PubMed

    Eduardo, Fernanda P; Mehnert, Dolores U; Monezi, Telma A; Zezell, Denise M; Schubert, Mark M; Eduardo, Carlos P; Marques, Márcia M

    2007-04-01

    Little is known about the intracellular response of epithelial cells to phototherapy. The aim of this in vitro study was to analyze the effect of phototherapy with low-energy lasers with different wavelengths and powers on cultured epithelial cell growth under different nutritional conditions. Epithelial cell cultures (Vero cell line) grown in nutritional deficit in culture medium supplemented with 2% fetal bovine serum (FBS) were irradiated with low-energy laser from one to three times with a GaAlAs laser (660 nm) and InGaAlP (780 nm), 40 and 70 mW, respectively, with 3 or 5 J/cm2. Cell growth was indirectly assessed by measuring the cell mitochondrial activity. Nonirradiated cell cultures grown in nutritional regular medium supplemented with 10% FBS produced higher cell growth than all cultures grown in nutritional deficit irradiated or not. The overall cell growth of cultures grown under nutritionally deficit conditions was significantly improved especially when irradiated with 780 nm for three times. Phototherapy with the laser parameters tested increases epithelial cell growth rate for cells stressed by growth under nutritionally deficient states. This cell growth improvement is directly proportional to the number of irradiations; however, was not enough to reach the full cell growth potential rate of Vero epithelial cell line observed when growing under nutritional regular condition. (c) 2007 Wiley-Liss, Inc.

  3. The beta-agonist isoproterenol attenuates EGF-stimulated wound closure in human airway epithelial cells.

    PubMed

    Schnackenberg, Bradley J; Jones, Stacie M; Pate, Crystal; Shank, Brian; Sessions, Laura; Pittman, Laura M; Cornett, Lawrence E; Kurten, Richard C

    2006-03-01

    Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although beta-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that beta-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the beta-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing beta2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways.

  4. 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

  5. Epithelial-mesenchymal transition as a fundamental underlying pathogenic process in COPD airways: fibrosis, remodeling and cancer.

    PubMed

    Nowrin, Kaosia; Sohal, Sukhwinder Singh; Peterson, Gregory; Patel, Rahul; Walters, Eugene Haydn

    2014-10-01

    Chronic obstructive pulmonary disease (COPD) is a complex condition, frequently with a mix of airway and lung parenchymal damage. However, the earliest changes are in the small airways, where most of the airflow limitation occurs. The pathology of small airway damage seems to be wall fibrosis and obliteration, but the whole airway is involved in a 'field effect'. Our novel observations on active epithelial-mesenchymal transition (EMT) in the airways of smokers, particularly in those with COPD, are changing the understanding of this airway pathology and the aetiology of COPD. EMT involves a cascade of regulatory changes that destabilise the epithelium with a motile and mesenchymal epithelial cell phenotype emerging. One important manifestation of EMT activity involves up-regulation of specific key transcription factors (TFs), such as Smads, Twist, and β-catenin. Such TFs can be used as EMT biomarkers, in recognisable patterns reflecting the potential major drivers of the process; for example, TGFβ, Wnt, and integrin-linked kinase systems. Thus, understanding the relative changes in TF activity during EMT may provide rich information on the mechanisms driving this whole process, and how they may change over time and with therapy. We have sought to review the current literature on EMT and the relative expression of specific TF activity, to define the networks likely to be involved in a similar process in the airways of patients with smoking-related COPD.

  6. Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins.

    PubMed

    Francin-Allami, Mathilde; Merah, Kahina; Albenne, Cécile; Rogniaux, Hélène; Pavlovic, Marija; Lollier, Virginie; Sibout, Richard; Guillon, Fabienne; Jamet, Elisabeth; Larré, Colette

    2015-07-01

    Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.

  7. Regulation of extracellular matrix remodeling and cell fate determination by matrix metalloproteinase stromelysin-3 during thyroid hormone-dependent post-embryonic development.

    PubMed

    Shi, Yun-Bo; Fu, Liezhen; Hasebe, Takashi; Ishizuya-Oka, Atsuko

    2007-12-01

    Interactions between cells and extracellular matrix (ECM), in particular the basement membrane (BM), are fundamentally important for the regulation of a wide variety of physiological and pathological processes. Matrix metalloproteinases (MMP) play critical roles in ECM remodeling and/or regulation of cell-ECM interactions because of their ability to cleave protein components of the ECM. Of particular interest among MMP is stromelysin-3 (ST3), which was first isolated from a human breast cancer and also shown to be correlated with apoptosis during development and invasion of tumor cells in mammals. We have been using intestinal remodeling during thyroid hormone (TH)-dependent amphibian metamorphosis as a model to study the role of ST3 during post-embryonic tissue remodeling and organ development in vertebrates. This process involves complete degeneration of the tadpole or larval epithelium through apoptosis and de novo development of the adult epithelium. Here, we will first summarize expression studies by us and others showing a tight spatial and temporal correlation of the expression of ST3 mRNA and protein with larval cell death and adult tissue development. We will then review in vitro and in vivo data supporting a critical role of ST3 in TH-induced larval epithelial cell death and ECM remodeling. We will further discuss the potential mechanisms of ST3 function during metamorphosis and its broader implications.

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

    PubMed Central

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

    2016-01-01

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

  9. Gene Regulation by Retinoid Receptors in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2002-10-01

    Hamann , P. Jenti, B. Imhof, and D. Vestweber. 1993. A death and tissue remodeling during mouse mammary gland involution. De- monoclonal antibody...Cell Growth Differ 10:49-59. Kato J-Y, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ . 1993. Snowden AW, Perkins ND. 1998. Cell cycle regulation of the

  10. 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.

  11. HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

    PubMed Central

    Tugizov, Sharof M.; Herrera, Rossana; Veluppillai, Piri; Greenspan, Deborah; Soros, Vanessa; Greene, Warner C.; Levy, Jay A.; Palefsky, Joel M.

    2010-01-01

    Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission. PMID:21056450

  12. Epithelial cells are active participants in vocal fold wound healing: an in vivo animal model of injury.

    PubMed

    Leydon, Ciara; Imaizumi, Mitsuyoshi; Bartlett, Rebecca S; Wang, Sarah F; Thibeault, Susan L

    2014-01-01

    Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.

  13. Androgen-Dependent Sertoli Cell Tight Junction Remodeling Is Mediated by Multiple Tight Junction Components

    PubMed Central

    Chakraborty, Papia; William Buaas, F.; Sharma, Manju; Smith, Benjamin E.; Greenlee, Anne R.; Eacker, Stephen M.

    2014-01-01

    Sertoli cell tight junctions (SCTJs) of the seminiferous epithelium create a specialized microenvironment in the testis to aid differentiation of spermatocytes and spermatids from spermatogonial stem cells. SCTJs must be chronically broken and rebuilt with high fidelity to allow the transmigration of preleptotene spermatocytes from the basal to adluminal epithelial compartment. Impairment of androgen signaling in Sertoli cells perturbs SCTJ remodeling. Claudin (CLDN) 3, a tight junction component under androgen regulation, localizes to newly forming SCTJs and is absent in Sertoli cell androgen receptor knockout (SCARKO) mice. We show here that Cldn3-null mice do not phenocopy SCARKO mice: Cldn3−/− mice are fertile, show uninterrupted spermatogenesis, and exhibit fully functional SCTJs based on imaging and small molecule tracer analyses, suggesting that other androgen-regulated genes must contribute to the SCARKO phenotype. To further investigate the SCTJ phenotype observed in SCARKO mutants, we generated a new SCARKO model and extensively analyzed the expression of other tight junction components. In addition to Cldn3, we identified altered expression of several other SCTJ molecules, including down-regulation of Cldn13 and a noncanonical tight junction protein 2 isoform (Tjp2iso3). Chromatin immunoprecipitation was used to demonstrate direct androgen receptor binding to regions of these target genes. Furthermore, we demonstrated that CLDN13 is a constituent of SCTJs and that TJP2iso3 colocalizes with tricellulin, a constituent of tricellular junctions, underscoring the importance of androgen signaling in the regulation of both bicellular and tricellular Sertoli cell tight junctions. PMID:24825397

  14. Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

    PubMed Central

    Bonuccelli, Gloria; Molchansky, Alex; Capozza, Franco; Witkiewicz, Agnieszka K; Birbe, Ruth C; Howell, Anthony; Pestell, Richard G; Whitaker-Menezes, Diana; Sotgia, Federica

    2011-01-01

    We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism. PMID:21646868

  15. 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.

  16. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  17. Semaphorin 3A Contributes to Distal Pulmonary Epithelial Cell Differentiation and Lung Morphogenesis

    PubMed Central

    Becker, Patrice M.; Tran, Tracy S.; Delannoy, Michael J.; He, Chaoxia; Shannon, John M.; McGrath-Morrow, Sharon

    2011-01-01

    Rationale Semaphorin 3A (Sema3A) is a neural guidance cue that also mediates cell migration, proliferation and apoptosis, and inhibits branching morphogenesis. Because we have shown that genetic deletion of neuropilin-1, which encodes an obligatory Sema3A co-receptor, influences airspace remodeling in the smoke-exposed adult lung, we sought to determine whether genetic deletion of Sema3A altered distal lung structure. Methods To determine whether loss of Sema3A signaling influenced distal lung morphology, we compared pulmonary histology, distal epithelial cell morphology and maturation, and the balance between lung cell proliferation and death, in lungs from mice with a targeted genetic deletion of Sema3A (Sema3A-/-) and wild-type (Sema3A+/+) littermate controls. Results Genetic deletion of Sema3A resulted in significant perinatal lethality. At E17.5, lungs from Sema3A-/- mice had thickened septae and reduced airspace size. Distal lung epithelial cells had increased intracellular glycogen pools and small multivesicular and lamellar bodies with atypical ultrastructure, as well as reduced expression of type I alveolar epithelial cell markers. Alveolarization was markedly attenuated in lungs from the rare Sema3A-/- mice that survived the immediate perinatal period. Furthermore, Sema3A deletion was linked with enhanced postnatal alveolar septal cell death. Conclusions These data suggest that Sema3A modulates distal pulmonary epithelial cell development and alveolar septation. Defining how Sema3A influences structural plasticity of the developing lung is a critical first step for determining if this pathway can be exploited to develop innovative strategies for repair after acute or chronic lung injury. PMID:22096573

  18. Mitochondrial biogenesis and proteome remodeling promotes one carbon metabolism for T cell activation

    PubMed Central

    Ron-Harel, Noga; Santos, Daniel; Ghergurovich, Jonathan M.; Sage, Peter T.; Reddy, Anita; Lovitch, Scott B.; Dephoure, Noah; Satterstrom, F. Kyle; Sheffer, Michal; Spinelli, Jessica B.; Gygi, Steven; Rabinowitz, Joshua D.; Sharpe, Arlene H.; Haigis, Marcia C.

    2017-01-01

    Summary Naïve T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naïve CD4+ T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture, and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one carbon metabolism that is critical for T cell activation and survival. PMID:27411012

  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. Culture, Immortalization, and Characterization of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Liu, Shaohui; Hatton, Mark P.; Khandelwal, Payal

    2010-01-01

    Purpose. Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. Methods. Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. Results. It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. Conclusions. The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized. PMID:20335607

  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. Cell volume regulation in epithelial physiology and cancer

    PubMed Central

    Pedersen, Stine F.; Hoffmann, Else K.; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed. PMID:24009588

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

    PubMed Central

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

    2016-01-01

    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. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  5. Expression of growth factor and receptor mRNAs in skin epithelial cells following acute cutaneous injury.

    PubMed Central

    Antoniades, H. N.; Galanopoulos, T.; Neville-Golden, J.; Kiritsy, C. P.; Lynch, S. E.

    1993-01-01

    We report that acute injury induces the expression of selective growth factor and growth factor receptors in the epithelial cells of the wounded tissue. In situ hybridization analysis of skin biopsy specimens obtained after cutaneous injury in swine demonstrated the induction of the expression of transforming growth factor-alpha, its receptor, epidermal growth factor-R, acidic fibroblast growth factor, and basic fibroblast growth factor messenger RNAs in the skin epithelial cells of the wounded tissue. There was no significant expression in the epithelial cells of control, uninjured tissues. The expression levels were maximal during the period of active tissue repair (1 to 5 days after injury) and were totally suppressed upon the healing of the wounded tissues. In contrast, insulinlike growth factor-I, (IGF-I), IGF-I receptor, and IGF-II receptor messenger RNAs were expressed in the epithelial cells of both the control, uninjured tissues and in tissue specimens obtained after injury. There was no significant expression of IGF-II messenger RNA in the epithelial cells before or after injury. It seems that injury induces the coordinated expression of selective growth factor and growth factor receptor genes whose products contribute to the regulation of the complex processes involved in tissue repair and remodeling. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8386442

  6. Ion pump sorting in polarized renal epithelial cells.

    PubMed

    Caplan, M J

    2001-08-01

    The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

  7. Secretion of alpha 1-antitrypsin by alveolar epithelial cells.

    PubMed

    Venembre, P; Boutten, A; Seta, N; Dehoux, M S; Crestani, B; Aubier, M; Durand, G

    1994-06-13

    We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce alpha 1-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.

  8. Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis.

    PubMed

    Gifford, Gail B; Demitrack, Elise S; Keeley, Theresa M; Tam, Andrew; La Cunza, Nilsa; Dedhia, Priya H; Spence, Jason R; Simeone, Diane M; Saotome, Ichiko; Louvi, Angeliki; Siebel, Christian W; Samuelson, Linda C

    2017-06-01

    We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue. Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors. Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth. Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  9. Metformin inhibits the proliferation of benign prostatic epithelial cells

    PubMed Central

    Ge, Rongbin; Li, Jijun; Johnson, Cameron W.; Rassoulian, Cyrus; Olumi, Aria F.

    2017-01-01

    Objective Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the

  10. 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

  11. 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.

  12. Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance.

    PubMed

    Ene, Iuliana V; Walker, Louise A; Schiavone, Marion; Lee, Keunsook K; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A R; Munro, Carol A; Brown, Alistair J P

    2015-07-28

    The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by

  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. 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.

  15. 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.

  16. Ezrin Is an Effector of Hepatocyte Growth Factor–mediated Migration and Morphogenesis in Epithelial Cells

    PubMed Central

    Crepaldi, Tiziana; Gautreau, Alexis; Comoglio, Paolo M.; Louvard, Daniel; Arpin, Monique

    1997-01-01

    The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane–cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling. PMID:9230083

  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.

  18. How Shigella Utilizes Ca2+ Jagged Edge Signals during Invasion of Epithelial Cells

    PubMed Central

    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 Ca2+ responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca2+ 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 Ca2+ overload. In this review, we will focus on the role of Ca2+ responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

  19. Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro

    PubMed Central

    Zheng, Dahai; Soh, Boon-Seng; Yin, Lu; Hu, Guangan; Chen, Qingfeng; Choi, Hyungwon; Han, Jongyoon; Chow, Vincent T. K.; Chen, Jianzhu

    2017-01-01

    Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells’ capacity to differentiate into alveolar epithelial cells at the single-cell level. PMID:28128362

  20. Chromatin Remodeling, Cell Proliferation and Cell Death in Valproic Acid-Treated HeLa Cells

    PubMed Central

    Felisbino, Marina Barreto; Tamashiro, Wirla M. S. C.; Mello, Maria Luiza S.

    2011-01-01

    Background Valproic acid (VPA) is a potent anticonvulsant that inhibits histone deacetylases. Because of this inhibitory action, we investigated whether VPA would affect chromatin supraorganization, mitotic indices and the frequency of chromosome abnormalities and cell death in HeLa cells. Methodology/Principal Findings Image analysis was performed by scanning microspectrophotometry for cells cultivated for 24 h, treated with 0.05, 0.5 or 1.0 mM VPA for 1–24 h, and subjected to the Feulgen reaction. TSA-treated cells were used as a predictable positive control. DNA fragmentation was investigated with the TUNEL assay. Chromatin decondensation was demonstrated under TSA and all VPA treatments, but no changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found with the VPA treatment conditions mentioned above, although decreased mitotic indices were detected under higher VPA concentration and longer exposure time. The frequency of DNA fragmentation identified with the TUNEL assay in HeLa cells increased after a 24-h VPA treatment, although this fragmentation occurred much earlier after treatment with TSA. Conclusions/Significance The inhibition of histone deacetylases by VPA induces chromatin remodeling in HeLa cells, which suggests an association to altered gene expression. Under VPA doses close to the therapeutic antiepileptic plasma range no changes in cell proliferation or chromosome abnormalities are elicited. The DNA fragmentation results indicate that a longer exposure to VPA or a higher VPA concentration is required for the induction of cell death. PMID:22206001

  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. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

    PubMed Central

    Hong, Deli; Messier, Terri L.; Tye, Coralee E.; Dobson, Jason R.; Fritz, Andrew J.; Sikora, Kenneth R.; Browne, Gillian; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

    2017-01-01

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression. PMID:28407681

  3. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition.

    PubMed

    Hong, Deli; Messier, Terri L; Tye, Coralee E; Dobson, Jason R; Fritz, Andrew J; Sikora, Kenneth R; Browne, Gillian; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2017-03-14

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

  4. Transcytotic passage of albumin through lens epithelial cells.

    PubMed

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

    2007-03-01

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

  5. 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.

  6. Anatomical location and culture of equine corneal epithelial stem cells.

    PubMed

    Moriyama, Hidekazu; Kasashima, Yoshinori; Kuwano, Atsutoshi; Wada, Shinya

    2014-03-01

    To identify morphologically the locations of equine corneal epithelial stem cells (CESCs) and to culture these cells. We studied the eyes of 12 adult thoroughbred horses. Eye tissues were immunostained for two positive stem cell markers (p63, CK14) and one negative marker (CK3) to identify the locations of CESCs, so we could compare their immunostaining patterns with those of human stem cells previously reported. We compared the proliferation rates and morphological features of epithelial cells isolated from the corneal limbus and central cornea. Undifferentiated cells expressing the same immunostaining pattern as human CESCs were present in the equine corneal limbus. Cultured epithelial cells isolated from the limbus expressed the same immunostaining pattern that CESCs show histologically, but cells isolated from the central cornea did not proliferate and could not be evaluated. Equine CESCs were localized in the epithelial basal layer of the corneal limbus, where melanocytes reside. They could be cultured without loss of their undifferentiated nature. When collecting such stem cells, it may be useful to harvest and culture corneal epithelial tissues in the limbus where melanocytes serve as an indicator of the collecting area. © 2013 American College of Veterinary Ophthalmologists.

  7. 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.

  8. 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.

  9. 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.

  10. Epithelial to Mesenchymal Transition of Mesothelial Cells in Tuberculous Pleurisy

    PubMed Central

    Kim, Changhwan; Park, Sung-Hoon; Hwang, Yong Il; Jang, Seung Hun; Kim, Cheol Hong; Jung, Ki-Suck; Min, Kwangseon; Lee, Jae Woong; Jang, Young Sook

    2011-01-01

    Purpose Tuberculous pleurisy is the most frequent extrapulmonary manifestation of tuberculosis. In spite of adequate treatment, pleural fibrosis is a common complication, but the mechanism has not been elucidated. This study is to determine whether epithelial to mesenchymal transition (EMT) of mesothelial cells occurs in tuberculous pleurisy. Materials and Methods Normal pleural mesothelial cells, isolated from irrigation fluids during operations for primary spontaneous pneumothorax, were characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). These cells were treated in vitro with various cytokines, which were produced in the effluents of tuberculous pleurisy. The isolated cells from the effluents of tuberculous pleurisy were analyzed by immunofluorescence and RT-PCR analysis. Results The isolated cells from the irrigation fluid of primary spontaneous pneumothorax had epithelial characteristics. These cells, with transforming growth factor-β1 and/or interleukin-1β treatment, underwent phenotypic transition from epithelial to mesenchymal cells, with the loss of epithelial morphology and reduction in cytokeratin and E-cadherin expression. Effluent analysis from tuberculous pleurisy using immunofluorescence and RT-PCR demonstrated two phenotypes that showed mesenchymal characteristics and both epithelial & mesencymal characteristics. Conclusion Our results suggest that pleural mesothelial cells in tuberculous pleurisy have been implicated in pleural fibrosis through EMT. PMID:21155035

  11. Epithelial cell guidance by self-generated EGF gradients†

    PubMed Central

    Scherber, Cally; Aranyosi, Alexander J.; Kulemann, Birte; Thayer, Sarah P.; Toner, Mehmet; Iliopoulos, Othon

    2012-01-01

    Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing. PMID:22314635

  12. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  13. Starved epithelial cells uptake extracellular matrix for survival

    PubMed Central

    Muranen, Taru; Iwanicki, Marcin P.; Curry, Natasha L.; Hwang, Julie; DuBois, Cory D.; Coloff, Jonathan L.; Hitchcock, Daniel S.; Clish, Clary B.; Brugge, Joan S.; Kalaany, Nada Y.

    2017-01-01

    Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition. PMID:28071763

  14. 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.

  15. 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.

  16. 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.

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

    NASA Astrophysics Data System (ADS)

    Yang, C. H.; Craise, L. M.

    1994-10-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  18. 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

  19. Mitral valve leaflet remodelling during pregnancy: insights into cell-mediated recovery of tissue homeostasis.

    PubMed

    Rego, Bruno V; Wells, Sarah M; Lee, Chung-Hao; Sacks, Michael S

    2016-12-01

    Little is known about how valvular tissues grow and remodel in response to altered loading. In this work, we used the pregnancy state to represent a non-pathological cardiac volume overload that distends the mitral valve (MV), using both extant and new experimental data and a modified form of our MV structural constitutive model. We determined that there was an initial period of permanent set-like deformation where no remodelling occurs, followed by a remodelling phase that resulted in near-complete restoration of homeostatic tissue-level behaviour. In addition, we observed that changes in the underlying MV interstitial cell (MVIC) geometry closely paralleled the tissue-level remodelling events, undergoing an initial passive perturbation followed by a gradual recovery to the pre-pregnant state. Collectively, these results suggest that valvular remodelling is actively mediated by average MVIC deformations (i.e. not cycle to cycle, but over a period of weeks). Moreover, tissue-level remodelling is likely to be accomplished by serial and parallel additions of fibrillar material to restore the mean homeostatic fibre stress and MVIC geometries. This finding has significant implications in efforts to understand and predict MV growth and remodelling following such events as myocardial infarction and surgical repair, which also place the valve under altered loading conditions.

  20. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

  1. 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

  2. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

  3. 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.

  4. 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

  5. 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

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

    PubMed

    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.

  7. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    PubMed

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  8. The mast cell - B-cell axis in lung vascular remodeling and pulmonary hypertension.

    PubMed

    Breitling, Siegfried; Hui, Zhang; Zabini, Diana; Hu, Yijie; Hoffmann, Julia; Goldenberg, Neil M; Tabuchi, Arata; Buelow, Roland; Dos Santos, Claudia; Kuebler, Wolfgang Michael

    2017-02-24

    Over the past years, a critical role for the immune system and in particular, for mast cells, in the pathogenesis of pulmonary hypertension (PH) has emerged. However, the way in which mast cells promote PH is still poorly understood. Here, we investigated the mechanisms by which mast cells may contribute to PH, specifically focusing on the interaction between the innate and adaptive immune response and the role of B-cells and autoimmunity. Experiments were performed in Sprague Dawley rats and B-cell deficient JH-KO rats in the monocrotaline, sugen-hypoxia and the aortic banding model of PH. Hemodynamics, cell infiltration, IL-6 expression, and vascular remodeling were analyzed. Gene array analyses revealed constituents of immunoglobulins as most prominently regulated mast cell dependent genes in the lung in experimental PH. IL-6 was shown to link mast cells to B-cells, as a) IL-6 was upregulated and colocalized with mast cells and was reduced by mast cell stabilizers, and b) IL-6 or mast cell blockade reduced B-cells in lungs of monocrotaline-treated rats. A functional role for B-cells in PH was demonstrated, in that either blocking B-cells by an anti-CD20 antibody or B-cell deficiency in JH-KO rats attenuated right ventricular systolic pressure and vascular remodeling in experimental PH. We here identify a mast cell - B-cell axis driven by IL-6 as critical immune pathway in the pathophysiology of PH. Our results provide novel insights into the role of the immune system in PH, which may be therapeutically exploited by targeted immunotherapy.

  9. Epithelial-mesenchymal transitions of bile duct epithelial cells in primary hepatolithiasis.

    PubMed

    Zhao, Lijin; Yang, Rigao; Cheng, Long; Wang, Maijian; Jiang, Yan; Wang, Shuguang

    2010-07-01

    The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, alpha-catenin, alpha-SMA, vimentin, S100A4, TGF-beta1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and alpha-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, alpha-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-beta1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-beta1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.

  10. 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.

  11. Fibrin glue inhibits migration of ocular surface epithelial cells.

    PubMed

    Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

    2016-10-01

    PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

  12. Serratia marcescens internalization and replication in human bladder epithelial cells

    PubMed Central

    Hertle, Ralf; Schwarz, Heinz

    2004-01-01

    Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

  13. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    PubMed

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  14. Quantification of three-dimensional cell-mediated collagen remodeling using graph theory.

    PubMed

    Bilgin, Cemal Cagatay; Lund, Amanda W; Can, Ali; Plopper, George E; Yener, Bülent

    2010-09-30

    Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Definition of early metrics that are able to predict long-term functionality by linking engineered tissue structure to function is an important step toward optimizing biomaterials for the purposes of regenerative medicine.

  15. Effects of lipopolysaccharide, multiwalled carbon nanotubes, and the combination on lung alveolar epithelial cells

    PubMed Central

    Pacurari, M; May, I; Tchounwou, PB

    2016-01-01

    Multi-walled carbon nanotubes (MWCNT) have been shown to induce lung fibrosis in animal models, however the underlying molecular factors/mechanisms are still unclear. In this study, we investigated the effects of lipopolysaccharide (LPS), MWCNT, and the combination of LPS and MWCNT on the expression of matrix metalloproteinase-9 and metalloproteinase-12 (MMP-9, MMP-12), collagen 3A1 (Col3A1), and transforming growth factor beta (TGFβ) in alveolar epithelial A549 cells. MMPs are proteinases that degrade extracellular matrix and play a role in lung fibrosis. A549 cells were exposed to LPS (1 ng/ml), MWCNT (20 μg/ml), and the combination and analyzed for paracellular permeability, TGFβ, Col3A1, MMP-9, MMP-12, NF-κB activation, and cell migration by real-time PCR and immunofluorescence. LPS, the combination of LPS and MWCNT, and MWCNT only at the highest tested dose induced blue dextran extravation. LPS and MWCNT increased the expression of TGFβ and its downstream target gene Col3A, and MMP-9 and MMP-12 mRNA. MWCNT potently induced cell migration toward wound healing, whereas LPS slightly induced cell migration. Both, LPS and MWCNT, induced NF-κB nuclear translocation. Our results indicate that MWCNT activated alveolar epithelial cells to promote fibrogenesis, and that LPS differentially primes molecular factors involved in lung remodeling. These findings suggest a role of alveolar epithelial cells in fibrogenesis and also may aid in the design and development of tests for screening of fibrogenic agents. PMID:26880698

  16. Nivalenol and Deoxynivalenol Affect Rat Intestinal Epithelial Cells: A Concentration Related Study

    PubMed Central

    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 G0/G1 interphase and in the S phase, with a concomitant reduction in the fraction of cells in G2. 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

  17. 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.

  18. Biphenotypic surface epithelial cells in the gastrointestinal tube with mixed epithelial-myofibroblastic differentiation: a paradigm.

    PubMed

    Németh, István Balázs; Tiszlavicz, László

    2012-04-01

    Epithelial cells and myofibroblasts are well-characterized histomorphological elements of tissues. They are distinguished from one another on the basis of topography and of differences in cytokeratin (CK) and α-smooth muscle actin (SMA) expression. Certain epithelial cells exhibit CK / SMA co-expression. This study aimed to define the immunophenotypical characteristics of these biphenotypic cells with respect to cytodifferentiation (broad spectrum of CKs, SMA), cell-cell interaction (E-cadherin, adenomatous polyposis coli - APC, β-catenin), and cell survival (cyclooxygenase-2 - Cox-2). At the routine gastrointestinal pathology service of the Department of Pathology, University of Szeged, tissue samples were identified from instances of cervical inlet patch (n = 5), Barrett's esophagus (n=5), gastritis (n=5), fundic gland polyp (n=2), gastric neoplastic polyp (n=1), inflammatory bowel disease (n=5), and colonic neoplastic polyp (n=3). that contained epithelial cells expressing SMA. These biphenotypic cells were further immunophenotyped. Foregut-derived biphenotypic cells expressed CKs 7 and 20, while hindgut-derived biphenotypic cells expressed only CK 20. Subepithelial myofibroblasts adjacent to biphenotypic epithelium expressed Cox-2, SMA, and β-catenin, as did biphenotypic cells. Myofibroblasts, however, did not express CKs. In neoplastic polyps, APC expression weakened as cytologic atypism increased, while intermingled biphenotypic cells in neoplastic glands overexpressed APC, as did myofibroblasts beneath. CK subspecies expression in biphenotypic cells reflects embryonic development of the gastrointestinal tract. The immunophenotyping analysis addresses bidirectional (via transdifferentiation from epithelia into myofibroblasts or vice versa) formation of biphenotypic cells within damaged epithelium, a phenomenon that must be further analysed.

  19. 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.

  20. Polarized Distribution of IQGAP Proteins in Gastric Parietal Cells and Their Roles in Regulated Epithelial Cell Secretion

    PubMed Central

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

    2003-01-01

    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. PMID:12631726

  1. Serelaxin improves the therapeutic efficacy of RXFP1-expressing human amnion epithelial cells in experimental allergic airway disease.

    PubMed

    Royce, Simon G; Tominaga, Anna M; Shen, Matthew; Patel, Krupesh P; Huuskes, Brooke M; Lim, Rebecca; Ricardo, Sharon D; Samuel, Chrishan S

    2016-12-01

    Current asthma therapies primarily target airway inflammation (AI) and suppress episodes of airway hyperresponsiveness (AHR) but fail to treat airway remodelling (AWR), which can develop independently of AI and contribute to irreversible airway obstruction. The present study compared the anti-remodelling and therapeutic efficacy of human bone marrow-derived mesenchymal stem cells (MSCs) to that of human amnion epithelial stem cells (AECs) in the setting of chronic allergic airways disease (AAD), in the absence or presence of an anti-fibrotic (serelaxin; RLX). Female Balb/c mice subjected to the 9-week model of ovalbumin (OVA)-induced chronic AAD, were either vehicle-treated (OVA alone) or treated with MSCs or AECs alone [intranasally (i.n.)-administered with 1×10(6) cells once weekly], RLX alone (i.n.-administered with 0.8 mg/ml daily) or a combination of MSCs or AECs and RLX from weeks 9-11 (n=6/group). Measures of AI, AWR and AHR were then assessed. OVA alone exacerbated AI, epithelial damage/thickness, sub-epithelial extracellular matrix (ECM) and total collagen deposition, markers of collagen turnover and AHR compared with that in saline-treated counterparts (all P<0.01 compared with saline-treated controls). RLX or AECs (but not MSCs) alone normalized epithelial thickness and partially diminished the OVA-induced fibrosis and AHR by ∼40-50% (all P<0.05 compared with OVA alone). Furthermore, the combination treatments normalized epithelial thickness, measures of fibrosis and AHR to that in normal mice, and significantly decreased AI. Although AECs alone demonstrated greater protection against the AAD-induced AI, AWR and AHR, compared with that of MSCs alone, combining RLX with MSCs or AECs reversed airway fibrosis and AHR to an even greater extent. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  2. Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

    PubMed

    Becker, Stephen M; Cho, Kyou-Nam; Guo, Xiaoti; Fendig, Kirsten; Oosman, Mohammed N; Whitehead, Robert; Cohn, Steven M; Houpt, Eric R

    2010-03-01

    Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

  3. 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

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

    PubMed Central

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

    2015-01-01

    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, were measured. LTβR, 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 MMP-9 and ADAM-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 alpha-smooth muscle actin. Thus, LIGHT and LTαβ induce a distinct steroid-resistant inflammatory signature in airway epithelial cells via constitutively expressed LTβR. 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. PMID:26209626

  5. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells.

    PubMed

    Bosshardt, D D; Nanci, A

    1998-02-01

    After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

  6. D-4F, an apolipoprotein A-I mimetic, inhibits TGF-β1 induced epithelial-mesenchymal transition in human alveolar epithelial cell.

    PubMed

    You, Jia; Wang, Jintao; Xie, Linshen; Zhu, Chengwen; Xiong, Jingyuan

    2016-10-01

    Emerging evidences support that transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-β1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10ng/ml of TGF-β1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-β1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-β1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-β1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Mesothelin promotes cell proliferation in the remodeling of neonatal rat pancreas

    PubMed Central

    Yin, Dan-Dan; You, Liang-Hui; Yuan, Qing-Xin; Liang, Xiao-Di; Wang, Ning; Wang, Lin-Tao; Yuan, Li; Wang, Ke-Ming; De, Wei

    2014-01-01

    AIM: To investigate the effect of mesothelin in the remodeling of the endocrine pancreas in neonatal rats. METHODS: Overexpression or downregulation of mesothelin expression in INS-1 cells was carried out to investigate the effect of mesothelin during cell proliferation and cell apoptosis in vitro. Adenovirus-mediated RNA interference was performed to block mesothelin in vivo to directly assess the role of mesothelin in the remodeling of the endocrine pancreas in neonatal rats. RESULTS: Exogenous overexpression of mesothelin promoted cell proliferation, cell colony formation and enhanced cell resistance to apoptosis of INS-1 cells. Down-regulation of mesothelin made no difference in cell proliferation and apoptosis compared with that in the control group. After an injection of adenovirus-mesothelin, a significantly increased number of small islets appeared, and the expression of PCNA was decreased on day 7 and day 14 compared with the Ad-EGFP group. CONCLUSION: Mesothelin was able to promote β cell proliferation in the remodeling stage of neonatal rats. Mesothelin may have an important role in the remodeling of the endocrine pancreas in neonatal rats. PMID:24944479

  8. Mechanical remodeling of normally sized mammalian cells under a gravity vector.

    PubMed

    Zhang, Chen; Zhou, Lüwen; Zhang, Fan; Lü, Dongyuan; Li, Ning; Zheng, Lu; Xu, Yanhong; Li, Zhan; Sun, Shujin; Long, Mian

    2017-02-01

    Translocation of the dense nucleus along a gravity vector initiates mechanical remodeling of a cell, but the underlying mechanisms of cytoskeletal network and focal adhesion complex (FAC) reorganization in a mammalian cell remain unclear. We quantified the remodeling of an MC3T3-E1 cell placed in upward-, downward-, or edge-on-orientated substrate. Nucleus longitudinal translocation presents a high value in downward orientation at 24 h or in edge-on orientation at 72 h, which is consistent with orientation-dependent distribution of perinuclear actin stress fibers and vimentin cords. Redistribution of total FAC area and fractionized super mature adhesion number coordinates this dependence at short duration. This orientation-dependent remodeling is associated with nucleus flattering and lamin A/C phosphorylation. Actin depolymerization or Rho-associated protein kinase signaling inhibition abolishes the orientation dependence of nucleus translocation, whereas tubulin polymerization inhibition or vimentin disruption reserves the dependence. A biomechanical model is therefore proposed for integrating the mechanosensing of nucleus translocation with cytoskeletal remodeling and FAC reorganization induced by a gravity vector.-Zhang, C., Zhou, L., Zhang, F., Lü, D., Li, N., Zheng, L., Xu, Y., Li, Z., Sun, S., Long, M. Mechanical remodeling of normally sized mammalian cells under a gravity vector. © FASEB.

  9. 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

  10. Effect of Helicobacter pylori on gastric epithelial cells

    PubMed Central

    Alzahrani, Shatha; Lina, Taslima T; Gonzalez, Jazmin; Pinchuk, Irina V; Beswick, Ellen J; Reyes, Victor E

    2014-01-01

    The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world’s population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori’s avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori. PMID:25278677

  11. 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...

  12. 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

  13. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

    Immortal Transformation of Cultured Human Mammary Epithelial Cells. Cellular Oncology, 26:248-251, 2004. Rodier , F., Kim, S-H., Nijjar, T., Yaswen, P...Promoter, Mol. Cell Biol.: 25:3923-3933, 2005. Goldstein, J, Rodier , F, Garbe, J, Stampfer, M, Campisi, J, Caspase-independent cytochrome c release is a

  14. 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...

  15. 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...

  16. 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

  17. 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...

  18. Atrial natriuretic peptide: A novel mediator for TGF-β1-induced epithelial-mesenchymal transition in 16HBE-14o and A549 cells.

    PubMed

    Chu, Shuyuan; Zhang, Xiufeng; Sun, Yabing; Yu, Yuanyuan; Liang, Yaxi; Jiang, Ming; Huang, Jianwei; Ma, Libing

    2017-02-13

    Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-β1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA expressions were increased after induction by TGF-β1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and α-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased α-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with α-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-β1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-β1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.

  19. Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin

    PubMed Central

    Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.

    2011-01-01

    Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

  20. 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

  1. [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.

  2. Coronavirus entry and release in polarized epithelial cells: a review.

    PubMed

    Cong, Yingying; Ren, Xiaofeng

    2014-09-01

    Most coronaviruses cause respiratory or intestinal infections in their animal or human host. Hence, their interaction with polarized epithelial cells plays a critical role in the onset and outcome of infection. In this paper, we review the knowledge regarding the entry and release of coronaviruses, with particular emphasis on the severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. As these viruses approach the epithelial surfaces from the apical side, it is not surprising that coronavirus cell receptors are exposed primarily on the apical domain of polarized epithelial cells. With respect to release, all possibilities appear to occur. Thus, most coronaviruses exit through the apical surface, several through the basolateral one, although the Middle East respiratory syndrome coronavirus appears to use both sides. These observations help us understand the local or systematic spread of the infection within its host as well as the spread of the virus within the host population.

  3. 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

  4. Thyroid Hormone-Regulated Wnt5a/Ror2 Signaling Is Essential for Dedifferentiation of Larval Epithelial Cells into Adult Stem Cells in the Xenopus laevis Intestine

    PubMed Central

    Ishizuya-Oka, Atsuko; Kajita, Mitsuko; Hasebe, Takashi

    2014-01-01

    Background and Aims Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis. Methods/Findings Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation. Significance Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology. PMID:25211363

  5. 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.

  6. Intestinal epithelial cells and their role in innate mucosal immunity.

    PubMed

    Maldonado-Contreras, A L; McCormick, Beth A

    2011-01-01

    The mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts are covered by a layer of epithelial cells that are responsible for sensing and promoting a host immune response in order to establish the limits not only for commensal microorganisms but also for foreign organisms or particles. This is a remarkable task as the human body represents a composite of about 10 trillion human-self cells plus non-self cells from autochthonous or indigenous microbes that outnumber human cells 10:1. Hence, the homeostasis of epithelial cells that line mucosal surfaces relies on a fine-tuned immune system that patrols the boundaries between human and microbial cells. In the case of the intestine, the epithelial layer is composed of at least six epithelial cell lineages that act as a physiological barrier in addition to aiding digestion and the absorption of nutrients, water and electrolytes. In this review, we highlight the immense role of the intestinal epithelium in coordinating the mucosal innate immune response.

  7. 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

  8. EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

    PubMed Central

    Xiao, Jianqiao; Palefsky, Joel M.; Herrera, Rossana; Berline, Jennifer; Tugizov, Sharof M.

    2009-01-01

    We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with β1 and αv family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2low recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells. PMID:19394065

  9. MAPK pathway mediates epithelial-mesenchymal transition induced by paraquat in alveolar epithelial cells.

    PubMed

    Huang, Min; Wang, Ya-Peng; Zhu, Ling-Qin; Cai, Qian; Li, Hong-Hui; Yang, Hui-Fang

    2016-11-01

    Epithelial-mesenchymal transition (EMT) is believed to be involved in lung fibrosis process induced by paraquat (PQ); however, the molecular mechanism of this process has not been clearly established. The present study investigated the potential involvement of EMT after PQ poisoning. The expressions of EMT markers, such as E-cadherin and α-smooth muscle actin (α-SMA), at multiple time points after exposure to different concentrations of PQ were evaluated by western blot analysis. Following PQ treatment, EMT induction was observed under microscopy. Related fibrosis genes, including Matrix metalloproteinase 2 (MMP-2), Matrix metalloproteinase 9 (MMP-9), collagens type I (COL I), and type III (COL III), were also evaluated by measuring their mRNA levels using RT-PCR analysis. Signaling pathways were analyzed using selective pharmacological inhibitors for MAPK. Cell migration ability was evaluated by scratch wound and Transwell assays. The data showed that PQ-induced epithelial RLE-6NT cells to develop mesenchymal cell characteristics, as indicated by a significant decrease in the epithelial marker E-cadherin and a significant increase in the extracellular matrix (ECM) marker α-smooth muscle actin in a dose and time-dependent manner. Moreover, PQ-treated RLE-6NT cells had an EMT-like phenotype with elevated expression of MMP-2, MMP-9, and COL I and COL III and enhanced migration ability. Signal pathway analysis revealed that PQ-induced EMT led to ERK-1 and Smad2 phosphorylation through activation of the MAPK pathway. The results of the current study indicate that PQ-induced pulmonary fibrosis occurs via EMT, which is mediated by the MAPK pathway. This implies that the MAPK pathway is a promising therapeutic target in alveolar epithelial cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1407-1414, 2016. © 2015 Wiley Periodicals, Inc.

  10. 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

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

    PubMed

    Robinson, Benjamin K; Cortes, Ernesto; Rice, Alistair J; Sarper, Muge; Del Río Hernández, Armando

    2016-06-15

    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. © 2016. Published by The Company of Biologists Ltd.

  12. 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

  13. TRPV channels as thermosensory receptors in epithelial cells.

    PubMed

    Lee, Hyosang; Caterina, Michael J

    2005-10-01

    Temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels are critical contributors to normal pain and temperature sensation and therefore represent attractive targets for pain therapy. When these channels were first discovered, most attention was focused on their potential contributions to direct thermal activation of peripheral sensory neurons. However, recent anatomical, physiological, and behavioral studies have provided evidence that TRPV channels expressed in skin epithelial cells may also contribute to thermosensation in vitro and in vivo. Here, we review these studies and speculate on possible communication mechanisms from cutaneous epithelial cells to sensory neurons.

  14. Collaboration of epithelial cells with organized mucosal lymphoid tissues.

    PubMed

    Neutra, M R; Mantis, N J; Kraehenbuhl, J P

    2001-11-01

    Immune surveillance of mucosal surfaces requires the delivery of intact macromolecules and microorganisms across epithelial barriers to organized mucosal lymphoid tissues. Transport, processing and presentation of foreign antigens, as well as local induction and clonal expansion of antigen-specific effector lymphocytes, involves a close collaboration between organized lymphoid tissues and the specialized follicle-associated epithelium. M cells in the follicle-associated epithelium transport foreign macromolecules and microorganisms to antigen-presenting cells within and under the epithelial barrier. Determination of the earliest cellular interactions that occur in and under the follicle-associated epithelium could greatly facilitate the design of effective mucosal vaccines in the future.

  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. Feature quantification and abnormal detection on cervical squamous epithelial cells.

    PubMed

    Zhao, Mingzhu; Chen, Lei; Bian, Linjie; Zhang, Jianhua; Yao, Chunyan; Zhang, Jianwei

    2015-01-01

    Feature analysis and classification detection of abnormal cells from images for pathological analysis are an important issue for the realization of computer assisted disease diagnosis. This paper studies a method for cervical squamous epithelial cells. Based on cervical cytological classification standard and expert diagnostic experience, expressive descriptors are extracted according to morphology, color, and texture features of cervical scales epithelial cells. Further, quantificational descriptors related to cytopathology are derived as well, including morphological difference degree, cell hyperkeratosis, and deeply stained degree. The relationship between quantified value and pathological feature can be established by these descriptors. Finally, an effective method is proposed for detecting abnormal cells based on feature quantification. Integrated with clinical experience, the method can realize fast abnormal cell detection and preliminary cell classification.

  17. Osmosignaling and volume regulation in intestinal epithelial cells.

    PubMed

    Lim, Christina H; Bot, Alice G M; de Jonge, Hugo R; Tilly, Ben C

    2007-01-01

    Most cells have to perform their physiological functions under a variable osmotic stress, which, because of the relatively high permeability of the plasma membrane for water, may result in frequent alterations in cell size. Intestinal epithelial cells are especially prone to changes in cell volume because of their high capacity of salt and water transport and the high membrane expression of various nutrient transporters. Therefore, to avoid excessive shrinkage or swelling, enterocytes, like most cell types, have developed efficient mechanisms to maintain osmotic balance. This chapter reviews selected model systems that can be used to investigate cell volume regulation in intestinal epithelial cells, with emphasis on the regulatory volume decrease, and the methods available to study the compensatory redistribution of (organic) osmolytes. In addition, a brief summary is presented of the pathways involved in osmosensing and osmosignaling in the intestine.

  18. Matrix Metalloproteinases: Inflammatory Regulators of Cell Behaviors in Vascular Formation and Remodeling

    PubMed Central

    Chen, Qishan; Jin, Min; Yang, Feng; Zhu, Jianhua; Xiao, Qingzhong; Zhang, Li

    2013-01-01

    Abnormal angiogenesis and vascular remodeling contribute to pathogenesis of a number of disorders such as tumor, arthritis, atherosclerosis, restenosis, hypertension, and neurodegeneration. During angiogenesis and vascular remodeling, behaviors of stem/progenitor cells, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs) and its interaction with extracellular matrix (ECM) play a critical role in the processes. Matrix metalloproteinases (MMPs), well-known inflammatory mediators are a family of zinc-dependent proteolytic enzymes that degrade various components of ECM and non-ECM molecules mediating tissue remodeling in both physiological and pathological processes. MMPs including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, and MT1-MMP, are stimulated and activated by various stimuli in vascular tissues. Once activated, MMPs degrade ECM proteins or other related signal molecules to promote recruitment of stem/progenitor cells and facilitate migration and invasion of ECs and VSMCs. Moreover, vascular cell proliferation and apoptosis can also be regulated by MMPs via proteolytically cleaving and modulating bioactive molecules and relevant signaling pathways. Regarding the importance of vascular cells in abnormal angiogenesis and vascular remodeling, regulation of vascular cell behaviors through modulating expression and activation of MMPs shows therapeutic potential. PMID:23840100

  19. [Disodium cromoglycate--mast cell degranulation blocker in the process of tissue remodelation].

    PubMed

    Maxová, H; Vasilková, M; Tkaczyk, J; Vízek, M

    2010-01-01

    Disodium cromoglycate (DSCG) is a compound commonly used in the treatment of allergic diseases. The effect of DSCG is due to its ability to stabilize the mast cell membrane and to prevent release of histamine and inflammatory mediators. Mast cells are also an abundant source of tissue metalloproteinases, serine proteases and growth factors, which play an important role in the processes of the tissue remodeling. In this view the DSCG is a substance which allows us to study the mechanisms of the pulmonary vascular bed remodeling in the experimental animals exposed to chronic hypoxia and in a phase of the recovery from hypoxia.

  20. Nicotine transport in lung and non-lung epithelial cells.

    PubMed

    Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

    2017-11-01

    Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [(3)H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Switched-memory B cells remodel B cell receptors within secondary germinal centers

    PubMed Central

    Okitsu, Shinji L.; McHeyzer-Williams, Michael G.

    2015-01-01

    Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive B cell receptor (BCR) re-diversification, but underlying mechanisms remain unresolved. Here integrated specificity and function of individual memory B cell progeny reveal ongoing evolution of polyclonal antibody specificities through germinal center (GC) specific transcriptional activity. At the clonal and sub-clonal levels, single cell expression of Cd83 and Pol□ segregates the secondary GC transcriptional program into 4 stages that regulate divergent mechanisms of memory BCR evolution. These studies demonstrate that vaccine boosts re-activate a cyclic program of GC function in switched-memory B cells to remodel existing antibody specificities and enhance durable immune protection. PMID:25642821

  2. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

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

    PubMed

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

    2017-02-15

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

  4. Keratins are novel markers of renal epithelial cell injury.

    PubMed

    Djudjaj, Sonja; Papasotiriou, Marios; Bülow, Roman D; Wagnerova, Alexandra; Lindenmeyer, Maja T; Cohen, Clemens D; Strnad, Pavel; Goumenos, Dimitrios S; Floege, Jürgen; Boor, Peter

    2016-04-01

    Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  5. Microarray analysis of human epithelial cell responses to bacterial interaction.

    PubMed

    Mans, Jeffrey J; Lamont, Richard J; Handfield, Martin

    2006-09-01

    Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has--until recently--held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

  6. CB1 cannabinoid receptor-mediated neurite remodeling in mouse neuroblastoma N1E-115 cells.

    PubMed

    Zhou, D; Song, Z H

    2001-08-15

    The morphological remodeling of neuronal cells influences neurogenesis and brain functions. We hypothesize that psychoactive and neurotoxic effects of cannabinoids may be mediated, at least in part, by their morphoregulatory activities. In the present study, mouse neuroblastoma N1E-115 cells were used as an in vitro model to investigate cannabinoid-induced neurite remodeling effects and to identify the involvement of cannabinoid receptors in this neurite remodeling process. Using reverse transcription-polymerase chain reaction and immunofluorescence microscopy, the endogenously expressed CB1, but not CB2, cannabinoid receptors were detected in morphologically differentiated N1E-115 cells. Activation of these natively expressed CB1 cannabinoid receptors by cannabinoid agonist HU-210 led to a concentration-dependent inhibition of adenylate cyclase activity. Importantly, HU-210 treatment induced neurite retraction in a concentration-dependent manner. Pretreatment of N1E-115 cells with a CB1 antisense oligodeoxynucleotide (ODN) suppressed HU-210-induced inhibition of forskolin-stimulated cAMP accumulation, indicating that the knocking down of functional CB1 cannabinoid receptor expression was achieved. Antisense ODN pretreatment also abolished HU-210-induced neurite retraction, demonstrating the involvement of CB1 cannabinoid receptors in mediating the neurite remodeling effects of HU-210. In addition, reversing HU-210-induced intracellular cAMP declination by 8-Br-cAMP partially prevented HU-210-induced neurite retraction, indicating the involvement of cAMP-dependent signaling pathways in mediating the neurite remodeling function of CB1 cannabinoid receptors in N1E-115 cells. These data demonstrate that neurite remodeling is a newly discovered function of CB1 cannabinoid receptors. This morphoregulatory function of CB1 cannabinoid receptors might be a new mechanism that mediates the psychoactive and neurotoxic effects of cannabinoids in developing and adult brain.

  7. Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

    PubMed Central

    Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

    2015-01-01

    ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

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

    PubMed Central

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

    2013-01-01

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

  9. Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos.

    PubMed

    Tian, J; Song, J; Li, H; Yang, D; Li, X; Ouyang, H; Lai, L

    2012-08-01

    Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.

  10. 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

  11. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells

    PubMed Central

    Padilla-Nash, Hesed M.; McNeil, Nicole E.

    2013-01-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  12. Clonogenic assay allows for selection of a primitive mammary epithelial cell population in bovine.

    PubMed

    Martignani, Eugenio; Cravero, Diego; Miretti, Silvia; Accornero, Paolo; Baratta, Mario

    2015-11-01

    Adult mammary stem cells have been identified in several species including the bovine. They are responsible for the development of the gland and for cyclic remodeling during estrous cycles and pregnancy. Epithelial cell subpopulations exist within the mammary gland. We and others showed previously that the Colony Forming Cell (CFC) assay can be used to detect lineage-restricted mammary progenitors. We carried out CFCs with bovine mammary cells and manually separated colonies with specific morphologies associated with either a luminal or a myoepithelial phenotype. Expression of specific markers was assessed by immunocytochemistry or by flow cytometry to confirm that the manual separation resulted in isolation of phenotipically different cells. When transplanted in recipient immunodeficient mice, we found that only myoepithelial-like colonies gave rise to outgrowths that resembled bovine mammary alveoli, thus proving that adult stem cells were maintained during culture and segregated with myoepithelial cells. After recovery of the cells from the transplanted mice and subsequent progenitor content analysis, we found a tendency to detect a higher progenitor frequency when myoepithelial-like colonies were transplanted. We here demonstrate that bovine adult mammary stem cells can be sustained in short-term culture and that they can be enriched by manually selecting for basal-like morphology.

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

    PubMed

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

    2016-10-02

    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.

  14. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

    PubMed Central

    Juncadella, Ignacio J.; Kadl, Alexandra; Sharma, Ashish K.; Shim, Yun M.; Hochreiter-Hufford, Amelia; Borish, Larry; Ravichandran, Kodi S.

    2013-01-01

    Lung epithelial cells can influence immune responses to airway allergens1,2. Airway epithelial cells also undergo apoptosis after encountering environmental allergens3; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells1,4,5. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens. PMID:23235830

  15. Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Sca1(+) Progenitor Stem Cell Expansion.

    PubMed

    Fitzpatrick, Emma; Han, Xu; Liu, Weimin; Corcoran, Eoin; Burtenshaw, Denise; Alshamrani, Maryam; Morrow, David; Helt, Jay-Christian; Cahill, Paul A; Redmond, Eileen M

    2017-09-18

    Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate Ethanol (EtOH) on Sonic Hedgehog (SHh) signaling in regulating possible Sca1(+) progenitor stem cell involvement during pathologic arterial remodeling. Partial ligation or sham-operation of the left carotid artery was performed in transgenic Sca1-eGFP mice gavaged with or without 'daily moderate' EtOH. The EtOH group had reduced adventitial thickening and less neo-intimal formation, compared to ligated controls. There was expansion of eGFP expressing (i.e., Sca1(+) ) cells in remodeled vessels post-ligation (14d), especially in the neo-intima. Ethanol treatment reduced the number of Sca1(+) cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 (Ptch1) and Gli2, and RT-PCR of whole vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1 - eGFP mice with the SHh signaling inhibitor cyclopamine diminished hedgehog target gene expression, reduced the number of Sca1(+) cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1(+) adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. EtOH reduces SHh - responsive Sca1(+) progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1(+) progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Epithelial neoplasia in Drosophila entails switch to primitive cell states

    PubMed Central

    Khan, Sumbul J.; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P.; Harsh, Sneh; Pandey, Ravi K.; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-01-01

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl− clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl− clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl− clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl− clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl− clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of “cells-of-origin” in epithelial cancers, namely their propensity for switch to primitive cell states. PMID:23708122

  17. Epithelial neoplasia in Drosophila entails switch to primitive cell states.

    PubMed

    Khan, Sumbul J; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P; Harsh, Sneh; Pandey, Ravi K; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-06-11

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl(-) clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl(-) clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl(-) clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl(-) clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl(-) clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

  18. Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth

    PubMed Central

    Meyer, Nicole; Woidacki, Katja; Knöfler, Martin; Meinhardt, Gudrun; Nowak, Désirée; Velicky, Philipp; Pollheimer, Jürgen; Zenclussen, Ana C.

    2017-01-01

    Intrauterine growth restriction (IUGR) is caused by insufficient remodeling of spiral arteries (SAs). The mechanism underlying the relevance of natural killer cells (NKs) and mast cells (MCs) for SA remodeling and its effects on pregnancy outcome are not well understood. We show that NK depletion arrested SA remodeling without affecting pregnancy. MC depletion resulted in abnormally remodeled SAs and IUGR. Combined absence of NKs and MCs substantially affected SA remodeling and impaired fetal growth. We found that α-chymase mast cell protease (Mcpt) 5 mediates apoptosis of uterine smooth muscle cells, a key feature of SA remodeling. Additionally, we report a previously unknown source for Mcpt5: uterine (u) NKs. Mice with selective deletion of Mcpt5+ cells had un-remodeled SAs and growth-restricted progeny. The human α-chymase CMA1, phylogenetic homolog of Mcpt5, stimulated the ex vivo migration of human trophoblasts, a pre-requisite for SA remodeling. Our results show that chymases secreted by uMCs and uNKs are pivotal to the vascular changes required to support pregnancy. Understanding the mechanisms underlying pregnancy-induced vascular changes is essential for developing therapeutic options against pregnancy complications associated with poor vascular remodeling. PMID:28327604

  19. Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth.

    PubMed

    Meyer, Nicole; Woidacki, Katja; Knöfler, Martin; Meinhardt, Gudrun; Nowak, Désirée; Velicky, Philipp; Pollheimer, Jürgen; Zenclussen, Ana C

    2017-03-22

    Intrauterine growth restriction (IUGR) is caused by insufficient remodeling of spiral arteries (SAs). The mechanism underlying the relevance of natural killer cells (NKs) and mast cells (MCs) for SA remodeling and its effects on pregnancy outcome are not well understood. We show that NK depletion arrested SA remodeling without affecting pregnancy. MC depletion resulted in abnormally remodeled SAs and IUGR. Combined absence of NKs and MCs substantially affected SA remodeling and impaired fetal growth. We found that α-chymase mast cell protease (Mcpt) 5 mediates apoptosis of uterine smooth muscle cells, a key feature of SA remodeling. Additionally, we report a previously unknown source for Mcpt5: uterine (u) NKs. Mice with selective deletion of Mcpt5(+) cells had un-remodeled SAs and growth-restricted progeny. The human α-chymase CMA1, phylogenetic homolog of Mcpt5, stimulated the ex vivo migration of human trophoblasts, a pre-requisite for SA remodeling. Our results show that chymases secreted by uMCs and uNKs are pivotal to the vascular changes required to support pregnancy. Understanding the mechanisms underlying pregnancy-induced vascular changes is essential for developing therapeutic options against pregnancy complications associated with poor vascular remodeling.

  20. Experimental Myocardial Infarction Induces Altered Regulatory T Cell Hemostasis, and Adoptive Transfer Attenuates Subsequent Remodeling

    PubMed Central

    Sharir, Rinat; Semo, Jonathan; Shimoni, Sara; Ben-Mordechai, Tamar; Landa-Rouben, Natalie; Maysel-Auslender, Sofia; Shaish, Aviv; Entin–Meer, Michal; Keren, Gad; George, Jacob

    2014-01-01

    Background Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling. Methods and Results The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling. Conclusion Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling. PMID:25436994

  1. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion.

    PubMed

    Grieve, Adam G; Rabouille, Catherine

    2014-08-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main component of adherens junctions, has been shown to be essential for epithelial cell extrusion, but its mechanistic contribution remains unclear. Here, we provide clear evidence that cell extrusion can be driven by the cleavage of E-cad, both in a wild-type and an oncogenic environment. We first show that CDC42 activation in a single epithelial cell results in its efficient matrix metalloproteinase (MMP)-sensitive extrusion through MEK signalling activation and this is supported by E-cad cleavage. Second, using an engineered cleavable form of E-cad, we demonstrate that, by itself, truncation of extracellular E-cad at the plasma membrane promotes apical extrusion. We propose that extracellular cleavage of E-cad generates a rapid change in cell-cell adhesion that is sufficient to drive apical cell extrusion. Whereas in normal epithelia, extrusion is followed by apoptosis, when combined with active oncogenic signalling, it is coupled to cell proliferation.

  2. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

    PubMed

    Arafa, Emad; Bondzie, Philip A; Rezazadeh, Kobra; Meyer, Rosana D; Hartsough, Edward; Henderson, Joel M; Schwartz, John H; Chitalia, Vipul; Rahimi, Nader

    2015-10-01

    Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.

  3. 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.

  4. [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.

  5. Hyperoxia-induced signal transduction pathways in pulmonary epithelial cells

    PubMed Central

    Zaher, Tahereh E.; Miller, Edmund J.; Morrow, Dympna M. P.; Javdan, Mohammad; Mantell, Lin L.

    2007-01-01

    Mechanical ventilation with hyperoxia is necessary to treat critically ill patients. However, prolonged exposure to hyperoxia leads to the generation of excessive reactive oxygen species (ROS), which can cause acute inflammatory lung injury. One of the major effects of hyperoxia is the injury and death of pulmonary epithelium, which is accompanied by increased levels of pulmonary proinflammatory cytokines and excessive leukocyte infiltration. A thorough understanding of the signaling pathways leading to pulmonary epithelial cell injury/death may provide some insights into the pathogenesis of hyperoxia-induced acute inflammatory lung injury. This review focuses on epithelial responses to hyperoxia and some of the major factors regulating pathways to epithelial cell injury/death, and proinflammatory responses upon exposure to hyperoxia. We discuss in detail some of the most interesting players, such as, NF-κB, that can modulate both proinflammatory responses and cell injury/death of lung epithelial cells. A better appreciation for the functions of these factors will no doubt help us to delineate the pathways to hyperoxic cell death and proinflammatory responses. PMID:17349918

  6. Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

    PubMed Central

    Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C.; Hershenson, Marc B.

    2008-01-01

    Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function. PMID:18787220

  7. 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.

  8. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

    PubMed

    Yoshinaga, Tomoyo; Uwabe, Kenichiro; Naito, Shoichi; Higashino, Kenichi; Nakano, Toru; Numata, Yoshito; Kihara, Akio

    2016-01-01

    Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

  9. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells

    PubMed Central

    Yoshinaga, Tomoyo; Uwabe, Kenichiro; Naito, Shoichi; Higashino, Kenichi; Nakano, Toru; Numata, Yoshito

    2016-01-01

    Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis. PMID:27936102

  10. Stem cell-based growth, regeneration, and remodeling of the planarian intestine

    PubMed Central

    Forsthoefel, David J.; Park, Amanda E.; Newmark, Phillip A.

    2011-01-01

    Although some animals are capable of regenerating organs, the mechanisms by which this is achieved are poorly understood. In planarians, pluripotent somatic stem cells called neoblasts supply new cells for growth, replenish tissues in response to cellular turnover, and regenerate tissues after injury. For most tissues and organs, however, the spatiotemporal dynamics of stem cell differentiation and the fate of tissue that existed prior to injury have not been characterized systematically. Utilizing in vivo imaging and bromodeoxyuridine pulse-chase experiments, we have analyzed growth and regeneration of the planarian intestine, the organ responsible for digestion and nutrient distribution. During growth, we observe that new gut branches are added along the entire anteroposterior axis. We find that new enterocytes differentiate throughout the intestine rather than in specific growth zones, suggesting that branching morphogenesis is achieved primarily by remodeling of differentiated intestinal tissues. During regeneration, we also demonstrate a previously unappreciated degree of intestinal remodeling, in which pre-existing posterior gut tissue contributes extensively to the newly formed anterior gut, and vice versa. By contrast to growing animals, differentiation of new intestinal cells occurs at preferential locations, including within newly generated tissue (the blastema), and along pre-existing intestinal branches undergoing remodeling. Our results indicate that growth and regeneration of the planarian intestine are achieved by coordinated differentiation of stem cells and the remodeling of pre-existing tissues. Elucidation of the mechanisms by which these processes are integrated will be critical for understanding organogenesis in a post-embryonic context. PMID:21664348

  11. Stem cell-based growth, regeneration, and remodeling of the planarian intestine.

    PubMed

    Forsthoefel, David J; Park, Amanda E; Newmark, Phillip A

    2011-08-15

    Although some animals are capable of regenerating organs, the mechanisms by which this is achieved are poorly understood. In planarians, pluripotent somatic stem cells called neoblasts supply new cells for growth, replenish tissues in response to cellular turnover, and regenerate tissues after injury. For most tissues and organs, however, the spatiotemporal dynamics of stem cell differentiation and the fate of tissue that existed prior to injury have not been characterized systematically. Utilizing in vivo imaging and bromodeoxyuridine pulse-chase experiments, we have analyzed growth and regeneration of the planarian intestine, the organ responsible for digestion and nutrient distribution. During growth, we observe that new gut branches are added along the entire anteroposterior axis. We find that new enterocytes differentiate throughout the intestine rather than in specific growth zones, suggesting that branching morphogenesis is achieved primarily by remodeling of differentiated intestinal tissues. During regeneration, we also demonstrate a previously unappreciated degree of intestinal remodeling, in which pre-existing posterior gut tissue contributes extensively to the newly formed anterior gut, and vice versa. By contrast to growing animals, differentiation of new intestinal cells occurs at preferential locations, including within newly generated tissue (the blastema), and along pre-existing intestinal branches undergoing remodeling. Our results indicate that growth and regeneration of the planarian intestine are achieved by co-ordinated differentiation of stem cells and the remodeling of pre-existing tissues. Elucidation of the mechanisms by which these processes are integrated will be critical for understanding organogenesis in a post-embryonic context.

  12. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

    PubMed Central

    Gidfar, Sanaz; Milani, Farnoud Y.; Milani, Behrad Y.; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J.; Sagha, Hossein; Djalilian, Ali R.

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells. PMID:28054657

  13. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture.

    PubMed

    Gidfar, Sanaz; Milani, Farnoud Y; Milani, Behrad Y; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J; Sagha, Hossein; Djalilian, Ali R

    2017-01-05

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

  14. A conformational switch controls cell wall-remodelling enzymes required for bacterial cell division.

    PubMed

    Yang, Desirée C; Tan, Kemin; Joachimiak, Andrzej; Bernhardt, Thomas G

    2012-08-01

    Remodelling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyse PG are critical for these processes, but their activities must be tightly regulated to prevent the generation of lethal breaches in the PG matrix. Despite their importance, the mechanisms regulating PG hydrolase activity have remained elusive. Here we investigate the control of cell division hydrolases called amidases (AmiA, AmiB and AmiC) required for Escherichia coli cell division. Poorly regulated amiB mutants were isolated encoding lytic AmiB variants with elevated basal PG hydrolase activities in vitro. The structure of an AmiB orthologue was also solved, revealing that the active site of AmiB is occluded by a conserved alpha helix. Strikingly, most of the amino acid substitutions in the lytic AmiB variants mapped to this domain and are predicted to disrupt its interaction with the active site. Our results therefore support a model in which cell separation is stimulated by the reversible relief of amidase autoinhibition governed by conserved subcomplexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multienzyme PG-remodelling machines.

  15. 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.

  16. Cell associated urokinase activity and colonic epithelial cells in health and disease.

    PubMed Central

    Gibson, P R; van de Pol, E; Doe, W F

    1991-01-01

    It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA. PMID:1650741

  17. 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...

  18. 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...

  19. Metabolic cooperativity between epithelial cells and adipocytes of mice

    SciTech Connect

    Bartley, J.C.; Emerman, J.T.; Bissell, M.J.

    1981-01-01

    We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from (/sup 14/C)glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy. We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations.

  20. Mesenchymal Stromal Cell-Derived Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Acquisition of Epithelial Stem-Like Cell Properties in Ameloblastoma Epithelial Cells.

    PubMed

    Jiang, Chunmiao; Zhang, Qunzhou; Shanti, Rabie M; Shi, Shihong; Chang, Ting-Han; Carrasco, Lee; Alawi, Faizan; Le, Anh D

    2017-09-01

    Epithelial-mesenchymal transition (EMT), a biological process associated with cancer stem-like or cancer-initiating cell formation, contributes to the invasiveness, metastasis, drug resistance, and recurrence of the malignant tumors; it remains to be determined whether similar processes contribute to the pathogenesis and progression of ameloblastoma (AM), a benign but locally invasive odontogenic neoplasm. Here, we demonstrated that EMT- and stem cell-related genes were expressed in the epithelial islands of the most common histologic variant subtype, the follicular AM. Our results revealed elevated interleukin (IL)-6 signals that were differentially expressed in the stromal compartment of the follicular AM. To explore the stromal effect on tumor pathogenesis, we isolated and characterized both mesenchymal stromal cells (AM-MSCs) and epithelial cells (AM-EpiCs) from follicular AM and demonstrated that, in in vitro culture, AM-MSCs secreted a significantly higher level of IL-6 as compared to the counterpart AM-EpiCs. Furthermore, both in vitro and in vivo studies revealed that exogenous and AM-MSC-derived IL-6 induced the expression of EMT- and stem cell-related genes in AM-EpiCs, whereas such effects were significantly abrogated either by a specific inhibitor of STAT3 or ERK1/2, or by knockdown of Slug gene expression. These findings suggest that AM-MSC-derived IL-6 promotes tumor-stem like cell formation by inducing EMT process in AM-EpiCs through STAT3 and ERK1/2-mediated signaling pathways, implying a role in the etiology and progression of the benign but locally invasive neoplasm. Stem Cells 2017;35:2083-2094. © 2017 AlphaMed Press.

  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. 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.

  3. 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

  4. Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells.

    PubMed

    Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J

    2013-04-01

    Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high-resolution whole-slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. "Virtually digested" WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here. Copyright © 2012 American Association for the Study of Liver Diseases.

  5. Epithelial remodeling after partial topography-guided normalization and high-fluence short-duration crosslinking (Athens protocol): results up to 1 year.

    PubMed

    Kanellopoulos, Anastasios John; Asimellis, George

    2014-10-01

    To compare epithelial remodeling in keratoconic eyes that had photorefractive keratectomy and corneal collagen crosslinking (Athens protocol) with that in untreated keratoconic eyes and healthy eyes. Private clinical practice, Athens, Greece. Comparative case series. Fourier-domain anterior segment optical coherence tomography (AS-OCT) was used to obtain in vivo 3-dimensional epithelial thickness maps and center, superior, inferior, maximum, minimum, mean, midperipheral, and variability data. Group A comprised 175 treated keratoconic eyes (Athens protocol); Group B, 193 untreated keratoconic eyes; and Group C, 160 healthy eyes. The 1-year mean center epithelial thickness in Group A was 47.78 μm ± 7.36 (SD) (range 33 to 64 μm). At the first clinical visit, it was 52.09 ± 6.80 μm (range 36 to 72 μm) in Group B and 52.54 ± 3.23 μm (range 45 to 59 μm) in Group C. The mean thickness range in Group A at 1 year was -19.94 ± 7.21 μm (range -6 to -34 μm). It was -21.83 ± 12.07 μm (range -4 to -66 μm) in Group B and -6.86 ± 3.33 μm (range -3 to -29 μm) in Group C. The mean topographic thickness variability in Group A at 1 year was 4.64 ± 1.63 μm (range 1.6 to 8.1 μm) (P<.05). It was 5.77 ± 3.39 μm (range 1.3 to 17.8 μm) in Group B and 1.59 ± 0.79 μm (range 0.6 to 5.6 μm) in Group C. Anterior segment OCT indicated a thinner and more homogeneous remodeled epithelium in the keratoconic eyes treated using the Athens protocol. Dr. Kanellopoulos is a consultant to Alcon Surgical, Inc.; Wavelight Laser Technologie AG; Avedro, Inc.; and i-Optics Optikgeräte GmbH. Dr. Asimellis has no financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  6. Allogeneic Mesenchymal Precursor Cell Therapy to Limit Remodeling After Myocardial Infarction: The Effect of Cell Dosage

    PubMed Central

    Hamamoto, Hirotsugu; Gorman, Joseph H.; Ryan, Liam P.; Hinmon, Robin; Martens, Timothy P.; Schuster, Michael D.; Plappert, Theodore; Kiupel, Matti; St. John-Sutton, Martin G.; Itescu, Silviu; Gorman, Robert C.

    2011-01-01

    Background This experiment assessed the dose-dependent effect of a unique allogeneic STRO-3–positive mesenchymal precursor cell (MPC) on postinfarction left ventricular (LV) remodeling. The MPCs were administered in a manner that would simulate an off-the-self, early postinfarction, preventative approach to cardiac cell therapy in a sheep transmural myocardial infarct (MI) model. Methods Allogeneic MPCs were isolated from male crossbred sheep. Forty-six female sheep underwent coronary ligation to produce a transmural LV anteroapical infarction. One hour after infarction, the borderzone myocardium received an injection of 25, 75, 225, or 450 × 106 MPCs, or cell medium. Echocardiography was performed at 4 and 8 weeks after MI to quantify LV end-diastolic (LVEDV) and end-systolic volumes (LVESV), ejection fraction (EF), and infarct expansion. CD31 and smooth muscle actin (SMA) immunohistochemical staining was performed on infarct and borderzone specimens to quantify vascular density. Results Compared with controls, low-dose (25 and 75 × 106 cells) MPC treatment significantly attenuated infarct expansion and increases in LVEDV and LVESV. EF was improved at all cell doses. CD31 and SMA immunohistochemical staining demonstrated increased vascular density in the borderzone only at the lower cell doses. There was no evidence of myocardial regeneration within the infarct. Conclusion Allogeneic STRO-3 positive MPCs attenuate the remodeling response to transmural MI in a clinically relevant large-animal model. This effect is associated with vasculogenesis and arteriogenesis within the borderzone and infarct and is most pronounced at lower cell doses. PMID:19231391

  7. Structural anomalies of highly malignant respiratory tract epithelial cells

    SciTech Connect

    Manger, R.L.; Heckman, C.A.

    1982-11-01

    These studies were designed to determine whether cytostructural changes were related to malignancy and the loss of growth control in epithelial cells. Three highly malignant cell lines were derived from transplantable carcinomas of the respiratory tract and compared with three respiratory tract epithelial lines of negligible malignancy. Keratin cytoskeletons were visualized by indirect immunofluorescence staining, and sample photomicrographs representing each line were prepared. The criteria used in making the classifications to identify the features common to the highly malignant lines included the nonuniform spacing of cells in the field of view, the cell shape, and the presence of nonfluorescent areas in the lamellar cytoplasm. Since the nonuniformity of keratin distribution in the periphery of the malignant cells suggested a structural anomaly, the cell lines were also examined by scanning electron microscopy. Unlike cells from the lines of negligible malignancy, cells from two of the highly malignant lines showed thickenings in the subterminal portions of the lamellar cytoplasm. The results suggested that specific architectural changes at the cellular level might be linked to the process of