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1
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Boger KD, Sheridan AE, Ziegler AL, Blikslager AT. Mechanisms and modeling of wound repair in the intestinal epithelium. Tissue Barriers 2022; 11:2087454. [PMID: 35695206 PMCID: PMC10161961 DOI: 10.1080/21688370.2022.2087454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Abstract
The intestinal epithelial barrier is susceptible to injury from insults, such as ischemia or infectious disease. The epithelium's ability to repair wounded regions is critical to maintaining barrier integrity. Mechanisms of intestinal epithelial repair can be studied with models that recapitulate the in vivo environment. This review focuses on in vitro injury models and intestinal cell lines utilized in such systems. The formation of artificial wounds in a controlled environment allows for the exploration of reparative physiology in cell lines modeling diverse aspects of intestinal physiology. Specifically, the use of intestinal cell lines, IPEC-J2, Caco-2, T-84, HT-29, and IEC-6, to model intestinal epithelium is discussed. Understanding the unique systems available for creating intestinal injury and the differences in monolayers used for in vitro work is essential for designing studies that properly capture relevant physiology for the study of intestinal wound repair.
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Affiliation(s)
- Kasey D Boger
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Ana E Sheridan
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Amanda L Ziegler
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
| | - Anthony T Blikslager
- Comparative Medicine Institute, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
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2
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Oncel S, Basson MD. Gut homeostasis, injury, and healing: New therapeutic targets. World J Gastroenterol 2022; 28:1725-1750. [PMID: 35633906 PMCID: PMC9099196 DOI: 10.3748/wjg.v28.i17.1725] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 12/12/2021] [Accepted: 03/27/2022] [Indexed: 02/06/2023] Open
Abstract
The integrity of the gastrointestinal mucosa plays a crucial role in gut homeostasis, which depends upon the balance between mucosal injury by destructive factors and healing via protective factors. The persistence of noxious agents such as acid, pepsin, nonsteroidal anti-inflammatory drugs, or Helicobacter pylori breaks down the mucosal barrier and injury occurs. Depending upon the size and site of the wound, it is healed by complex and overlapping processes involving membrane resealing, cell spreading, purse-string contraction, restitution, differentiation, angiogenesis, and vasculogenesis, each modulated by extracellular regulators. Unfortunately, the gut does not always heal, leading to such pathology as peptic ulcers or inflammatory bowel disease. Currently available therapeutics such as proton pump inhibitors, histamine-2 receptor antagonists, sucralfate, 5-aminosalicylate, antibiotics, corticosteroids, and immunosuppressants all attempt to minimize or reduce injury to the gastrointestinal tract. More recent studies have focused on improving mucosal defense or directly promoting mucosal repair. Many investigations have sought to enhance mucosal defense by stimulating mucus secretion, mucosal blood flow, or tight junction function. Conversely, new attempts to directly promote mucosal repair target proteins that modulate cytoskeleton dynamics such as tubulin, talin, Ehm2, filamin-a, gelsolin, and flightless I or that proteins regulate focal adhesions dynamics such as focal adhesion kinase. This article summarizes the pathobiology of gastrointestinal mucosal healing and reviews potential new therapeutic targets.
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Affiliation(s)
- Sema Oncel
- Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
| | - Marc D Basson
- Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
- Department of Surgery, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
- Department of Pathology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, United States
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3
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Bilotta AJ, Ma C, Yang W, Yu Y, Yu Y, Zhao X, Zhou Z, Yao S, Dann SM, Cong Y. Propionate Enhances Cell Speed and Persistence to Promote Intestinal Epithelial Turnover and Repair. Cell Mol Gastroenterol Hepatol 2020; 11:1023-1044. [PMID: 33238220 PMCID: PMC7898181 DOI: 10.1016/j.jcmgh.2020.11.011] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 11/13/2020] [Accepted: 11/16/2020] [Indexed: 02/07/2023]
Abstract
BACKGROUND AND AIMS Gut bacteria-derived short-chain fatty acids (SCFAs) play crucial roles in the maintenance of intestinal homeostasis. However, how SCFAs regulate epithelial turnover and tissue repair remain incompletely understood. In this study, we investigated how the SCFA propionate regulates cell migration to promote epithelial renewal and repair. METHODS Mouse small intestinal epithelial cells (MSIE) and human Caco-2 cells were used to determine the effects of SCFAs on gene expression, proliferation, migration, and cell spreading in vitro. Video microscopy and single cell tracking were used to assess cell migration kinetically. 5-bromo-2'-deoxyuridine (BrdU) and hydroxyurea were used to assess the effects of SCFAs on migration in vivo. Lastly, an acute colitis model using dextran sulfate sodium (DSS) was used to examine the effects of SCFAs in vivo. RESULTS Using video microscopy and single cell tracking, we found that propionate promoted intestinal epithelial cell migration by enhancing cell spreading and polarization, which led to increases in both cell speed and persistence. This novel function of propionate was dependent on inhibition of class I histone deacetylases (HDAC) and GPR43 and required signal transducer and activator of transcription 3 (STAT3). Furthermore, using 5-bromo-2'-deoxyuridine (BrdU) and hydroxyurea in vivo, we found that propionate enhanced cell migration up the crypt-villus axis under homeostatic conditions, while also protecting against ulcer formation in experimental colitis. CONCLUSION Our results demonstrate a mechanism by which propionate stimulates cell migration in an HDAC inhibition, GPR43, and STAT3 dependent manner, and suggest that propionate plays an important role in epithelial migration independent of proliferation.
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Affiliation(s)
- Anthony J. Bilotta
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Chunyan Ma
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas,Department of Central Laboratory, Shandong Provincial Hospital Shandong First Medical University, Jinan, China
| | - Wenjing Yang
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Yanbo Yu
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Yu Yu
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Xiaojing Zhao
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Zheng Zhou
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Suxia Yao
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
| | - Sara M. Dann
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas
| | - Yingzi Cong
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas,Department of Pathology, University of Texas Medical Branch, Galveston, Texas,Correspondence Address correspondence to: Yingzi Cong, PhD, Department of Microbiology and Immunology, University of Texas Medical Branch, 4.142C Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1019. fax: (409) 772-5065.
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Baumann J, Huang SF, Gassmann M, Tsao CC, Ogunshola OO. Furin inhibition prevents hypoxic and TGFβ-mediated blood-brain barrier disruption. Exp Cell Res 2019; 383:111503. [PMID: 31336100 DOI: 10.1016/j.yexcr.2019.111503] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Revised: 06/25/2019] [Accepted: 07/15/2019] [Indexed: 12/12/2022]
Abstract
Hypoxic blood-brain barrier (BBB) dysfunction is a common feature of CNS diseases however mechanisms underlying barrier disturbance are still largely unknown. This study investigated the role of transforming growth factor β (TGFβ), a cytokine known to induce expression of the proprotein convertase Furin, in hypoxia-mediated barrier compromise. We show that exposure of brain endothelial cells (ECs) to hypoxia (1% O2) rapidly stimulates their migration. Additional exogenous TGFβ (0.4 nM) exposure potentiated this effect and increased Furin expression in a TGFβ type I receptor activin-like kinase 5 (ALK5) - dependent manner (prevented by 10 μM SB431542). Furin inhibition prevented hypoxia-induced EC migration and blocked TGFβ-induced potentiation suggesting existence of a feedback loop. TGFβ and Furin were also critical for hypoxia-induced BBB dysfunction. TGFβ treatment aggravated hypoxia-induced BBB permeability but ALK5 or Furin blockade reversed injury-induced permeability changes. Thus during insult Furin compromises endothelial integrity by mediating the effects of TGFβ. Targeting the Furin or ALK5 pathway may offer novel therapeutic strategies for improving BBB stability and CNS function during disease.
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Affiliation(s)
- Julia Baumann
- Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center Integrative Physiology (ZIHP), University of Zurich, Zurich, Switzerland
| | - Sheng-Fu Huang
- Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center Integrative Physiology (ZIHP), University of Zurich, Zurich, Switzerland
| | - Max Gassmann
- Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center Integrative Physiology (ZIHP), University of Zurich, Zurich, Switzerland
| | - Chih-Chieh Tsao
- Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center Integrative Physiology (ZIHP), University of Zurich, Zurich, Switzerland
| | - Omolara O Ogunshola
- Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center Integrative Physiology (ZIHP), University of Zurich, Zurich, Switzerland.
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5
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Friedrich M, Gerbeth L, Gerling M, Rosenthal R, Steiger K, Weidinger C, Keye J, Wu H, Schmidt F, Weichert W, Siegmund B, Glauben R. HDAC inhibitors promote intestinal epithelial regeneration via autocrine TGFβ1 signalling in inflammation. Mucosal Immunol 2019; 12:656-667. [PMID: 30674988 DOI: 10.1038/s41385-019-0135-7] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2018] [Revised: 12/29/2018] [Accepted: 01/02/2019] [Indexed: 02/04/2023]
Abstract
Intact epithelial barrier function is pivotal for maintaining intestinal homeostasis. Current therapeutic developments aim at restoring the epithelial barrier in inflammatory bowel disease. Histone deacetylase (HDAC) inhibitors are known to modulate immune responses and to ameliorate experimental colitis. However, their direct impact on epithelial barrier function and intestinal wound healing is unknown. In human and murine colonic epithelial cell lines, the presence of the HDAC inhibitors Givinostat and Vorinostat not only improved transepithelial electrical resistance under inflammatory conditions but also attenuated the passage of macromolecules across the epithelial monolayer. Givinostat treatment mediated an accelerated wound closure in scratch assays. In vivo, Givinostat treatment resulted in improved barrier recovery and epithelial wound healing in dextran sodium sulphate-stressed mice. Mechanistically, these regenerative effects could be linked to an increased secretion of transforming growth factor beta1 and interleukin 8, paralleled by differential expression of the tight junction proteins claudin-1, claudin-2 and occludin. Our data reveal a novel tissue regenerative property of the pan-HDAC inhibitors Givinostat and Vorinostat in intestinal inflammation, which may have beneficial implications by repurposing HDAC inhibitors for therapeutic strategies for inflammatory bowel disease.
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Affiliation(s)
- Marie Friedrich
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.,Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, Berlin, Germany
| | - Lorenz Gerbeth
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.,Department Medical Biotechnology, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany
| | - Marco Gerling
- Department of Biosciences and Nutrition, Center of Innovative Medicine, Karolinska Institutet, Huddinge, Sweden
| | - Rita Rosenthal
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany
| | - Katja Steiger
- Institute of Pathology, Technical University of Munich, Munich, Germany
| | - Carl Weidinger
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.,Clinical Scientist Program, Berlin Institute of Health (BIH), Berlin, Germany
| | - Jacqueline Keye
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.,Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, Berlin, Germany
| | - Hao Wu
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.,Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, Berlin, Germany
| | - Franziska Schmidt
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.,Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, Berlin, Germany
| | - Wilko Weichert
- Institute of Pathology, Technical University of Munich, Munich, Germany
| | - Britta Siegmund
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany
| | - Rainer Glauben
- Medical Department of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.
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Transcription factor CUX1 is required for intestinal epithelial wound healing and targets the VAV2-RAC1 Signalling complex. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2017; 1864:2347-2355. [DOI: 10.1016/j.bbamcr.2017.09.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Revised: 09/05/2017] [Accepted: 09/06/2017] [Indexed: 01/02/2023]
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Power KA, Lu JT, Monk JM, Lepp D, Wu W, Zhang C, Liu R, Tsao R, Robinson LE, Wood GA, Wolyn DJ. Purified rutin and rutin-rich asparagus attenuates disease severity and tissue damage following dextran sodium sulfate-induced colitis. Mol Nutr Food Res 2016; 60:2396-2412. [PMID: 27349947 DOI: 10.1002/mnfr.201500890] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2015] [Revised: 06/07/2016] [Accepted: 06/15/2016] [Indexed: 12/25/2022]
Abstract
SCOPE This study investigated the effects of cooked whole asparagus (ASP) versus its equivalent level of purified flavonoid glycoside, rutin (RUT), on dextran sodium sulfate (DSS)-induced colitis and subsequent colitis recovery in mice. METHODS AND RESULTS C57BL/6 male mice were fed an AIN-93G basal diet (BD), or BD supplemented with 2% cooked ASP or 0.025% RUT for 2 wks prior to and during colitis induction with 2% DSS in water for 7 days, followed by 5 days colitis recovery. In colitic mice, both ASP and RUT upregulated mediators of improved barrier integrity and enhanced mucosal injury repair (e.g. Muc1, IL-22, Rho-A, Rac1, and Reg3γ), increased the proportion of mouse survival, and improved disease activity index. RUT had the greatest effect in attenuating DSS-induced colonic damage indicated by increased crypt and goblet cell restitution, reduced colonic myeloperoxidase, as well as attenuated DSS-induced microbial dysbiosis (reduced Enterobacteriaceae and Bacteroides, and increased unassigned Clostridales, Oscillospira, Lactobacillus, and Bifidobacterium). CONCLUSION These findings demonstrate that dietary cooked ASP and its flavonoid glycoside, RUT, may be useful in attenuating colitis severity by modulating the colonic microenvironment resulting in reduced colonic inflammation, promotion of colonic mucosal injury repair, and attenuation of colitis-associated microbial dysbiosis.
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Affiliation(s)
- Krista A Power
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
- Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada
| | - Jenifer T Lu
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
- Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada
| | - Jennifer M Monk
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
- Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada
| | - Dion Lepp
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
| | - Wenqing Wu
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
| | - Claire Zhang
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
- Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada
| | - Ronghua Liu
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
| | - Rong Tsao
- Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada
| | - Lindsay E Robinson
- Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada
| | - Geoffrey A Wood
- Department of Pathobiology, University of Guelph, Guelph, ON, Canada
| | - David J Wolyn
- Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada
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8
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Ardizzone S, Bevivino G, Monteleone G. Mongersen, an oral Smad7 antisense oligonucleotide, in patients with active Crohn's disease. Therap Adv Gastroenterol 2016; 9:527-32. [PMID: 27366221 PMCID: PMC4913329 DOI: 10.1177/1756283x16636781] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
In Crohn's disease (CD), the tissue-damaging inflammation is sustained by defects of counter-regulatory mechanisms, which normally inhibit immune-inflammatory signals and promote repair of mucosal injury. In particular, in inflamed gut of CD patients there are elevated levels of Smad7, an intracellular protein that inhibits the function of transforming growth factor (TGF)-β1. Knockdown of Smad7 with a specific antisense oligonucleotide, named mongersen, restores TGF-β1 activity thus leading to suppression of inflammatory pathways and resolution of colitis in mice. Consistently, oral administration of mongersen to patients with active CD induces clinical remission. In this article, we review the available data supporting the pathogenic role of Smad7 in CD and discuss the results of recent phase I and II trials assessing the efficacy and safety of mongersen in CD patients.
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Affiliation(s)
- Sandro Ardizzone
- Gastroenterology Unit, Department of Biomedical and Clinical Sciences, ‘Luigi Sacco’ University Hospital, 20157 Milano, Italy
| | - Gerolamo Bevivino
- Department of Systems Medicine, University of Rome ‘Tor Vergata’, Via Montpellier, 1, 00133 Rome, Italy
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9
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Zundler S, Caioni M, Müller M, Strauch U, Kunst C, Woelfel G. K+ Channel Inhibition Differentially Regulates Migration of Intestinal Epithelial Cells in Inflamed vs. Non-Inflamed Conditions in a PI3K/Akt-Mediated Manner. PLoS One 2016; 11:e0147736. [PMID: 26824610 PMCID: PMC4732808 DOI: 10.1371/journal.pone.0147736] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2015] [Accepted: 01/07/2016] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Potassium channels have been shown to determine wound healing in different tissues, but their role in intestinal epithelial restitution--the rapid closure of superficial wounds by intestinal epithelial cells (IEC)--remains unclear. METHODS In this study, the regulation of IEC migration by potassium channel modulation was explored with and without additional epidermal growth factor (EGF) under baseline and interferon-γ (IFN-γ)-pretreated conditions in scratch assays and Boyden chamber assays using the intestinal epithelial cell lines IEC-18 and HT-29. To identify possibly involved subcellular pathways, Western Blot (WB)-analysis of ERK and Akt phosphorylation was conducted and PI3K and ERK inhibitors were used in scratch assays. Furthermore, mRNA-levels of the potassium channel KCNN4 were determined in IEC from patients suffering from inflammatory bowel diseases (IBD). RESULTS Inhibition of Ca(2+)-dependent potassium channels significantly increased intestinal epithelial restitution, which could not be further promoted by additional EGF. In contrast, inhibition of KCNN4 after pretreatment with IFN-γ led to decreased or unaffected migration. This effect was abolished by EGF. Changes in Akt, but not in ERK phosphorylation strongly correlated with these findings and PI3K but not ERK inhibition abrogated the effect of KCNN4 inhibition. Levels of KCNN4 mRNA were higher in samples from IBD patients compared with controls. CONCLUSIONS Taken together, we demonstrate that inhibition of KCNN4 differentially regulates IEC migration in IFN-γ-pretreated vs. non pretreated conditions. Moreover, our data propose that the PI3K signaling cascade is responsible for this differential regulation. Therefore, we present a cellular model that contributes new aspects to epithelial barrier dysfunction in chronic intestinal inflammation, resulting in propagation of inflammation and symptoms like ulcers or diarrhea.
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Affiliation(s)
- Sebastian Zundler
- Department of Internal Medicine I, Regensburg University Medical Center, Regensburg, Germany
- Department of Medicine 1, University of Erlangen-Nuremberg, Kussmaul Campus for Medical Research & Translational Research Center, Erlangen, Germany
| | - Massimiliano Caioni
- Department of Internal Medicine I, Regensburg University Medical Center, Regensburg, Germany
| | - Martina Müller
- Department of Internal Medicine I, Regensburg University Medical Center, Regensburg, Germany
| | - Ulrike Strauch
- Department of Internal Medicine I, Regensburg University Medical Center, Regensburg, Germany
| | - Claudia Kunst
- Department of Internal Medicine I, Regensburg University Medical Center, Regensburg, Germany
| | - Gisela Woelfel
- Department of Internal Medicine I, Regensburg University Medical Center, Regensburg, Germany
- * E-mail:
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10
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Costanzo M, Cesi V, Prete E, Negroni A, Palone F, Cucchiara S, Oliva S, Leter B, Stronati L. Krill oil reduces intestinal inflammation by improving epithelial integrity and impairing adherent-invasive Escherichia coli pathogenicity. Dig Liver Dis 2016; 48:34-42. [PMID: 26493628 DOI: 10.1016/j.dld.2015.09.012] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Revised: 09/03/2015] [Accepted: 09/19/2015] [Indexed: 12/11/2022]
Abstract
BACKGROUND Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage. AIMS We aimed at assessing the ability of krill oil to reduce intestinal inflammation by improving epithelial barrier integrity, increasing cell survival and reducing pathogenicity of adherent-invasive Escherichia coli. METHODS CACO2 and HT29 cells were exposed to cytomix (TNFα and IFNγ) to induce inflammation and co-exposed to cytomix and krill oil. E-cadherin, ZO-1 and F-actin levels were analyzed by immunofluorescence to assess barrier integrity. Scratch test was performed to measure wound healing. Cell survival was analyzed by flow cytometry. Adherent-invasive Escherichia coli LF82 was used for adhesion/invasion assay. RESULTS In inflamed cells E-cadherin and ZO-1 decreased, with loss of cell-cell adhesion, and F-actin polymerization increased stress fibres; krill oil restored initial conditions and improved wound healing, reduced bacterial adhesion/invasion in epithelial cells and survival within macrophages; krill oil reduced LF82-induced mRNA expression of pro-inflammatory cytokines. CONCLUSIONS Krill oil improves intestinal barrier integrity and epithelial restitution during inflammation and controls bacterial adhesion and invasion to epithelial cells. Thus, krill oil may represent an innovative tool to reduce intestinal inflammation.
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Affiliation(s)
| | - Vincenzo Cesi
- Department of Radiobiology and Human Health, ENEA, Rome, Italy
| | - Enrica Prete
- Department of Radiobiology and Human Health, ENEA, Rome, Italy
| | - Anna Negroni
- Department of Radiobiology and Human Health, ENEA, Rome, Italy
| | | | - Salvatore Cucchiara
- Department of Paediatrics and Infantile Neuropsychiatry, Paediatric Gastroenterology and Liver Unit, Sapienza University of Rome, Italy
| | - Salvatore Oliva
- Department of Paediatrics and Infantile Neuropsychiatry, Paediatric Gastroenterology and Liver Unit, Sapienza University of Rome, Italy
| | - Beatrice Leter
- Department of Paediatrics and Infantile Neuropsychiatry, Paediatric Gastroenterology and Liver Unit, Sapienza University of Rome, Italy
| | - Laura Stronati
- Department of Radiobiology and Human Health, ENEA, Rome, Italy.
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11
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Abstract
In Crohn's disease and ulcerative colitis, the tissue-damaging destructive immune response is sustained by defects of counterregulatory mechanisms, which normally attenuate inflammatory pathways and promote repair of mucosal injury. One such mechanism involves transforming growth factor-β1 (TGF-β1), a cytokine that is produced by multiple cell types and targets both immune and nonimmune cells. Both in vitro and in vivo studies strongly support the role of TGF-β1 as a negative regulator of mucosal inflammation and indicate that defective production/activity of this cytokine can lead to the development of or exacerbate colitis. Interestingly, in the inflamed intestine of patients with inflammatory bowel disease, TGF-β1 expression is upregulated but TGF-β1-mediated immunosuppression is markedly impaired because of high Smad7, an intracellular inhibitor of TGF-β1-associated signaling. Consistently, knockdown of Smad7 with a specific antisense oligonucleotide restores TGF-β1 activity, thus leading to decreased production of inflammatory cytokines in both colitic mice and inflammatory bowel disease patients and attenuates clinical activity in Crohn's disease patients. In this article, we review data supporting the role of Smad7 in the pathogenesis of inflammatory bowel disease and discuss whether inhibition of Smad7 is therapeutically useful in Crohn's disease and how the benefit/risk of such an intervention should be monitored in the patients.
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12
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Khan SA, Khan AM, Karim S, Kamal MA, Damanhouri GA, Mirza Z. Panacea seed "Nigella": A review focusing on regenerative effects for gastric ailments. Saudi J Biol Sci 2014; 23:542-53. [PMID: 27298589 PMCID: PMC4890198 DOI: 10.1016/j.sjbs.2014.10.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2014] [Revised: 10/01/2014] [Accepted: 10/02/2014] [Indexed: 02/07/2023] Open
Abstract
Nigella sativa (NS) or black cumin is a dark, thin, and crescent-shaped, seeded shrub belonging to the Ranunculaceae family commonly growing on Mediterranean coasts in Saudi Arabia, northern Africa and Asia. They have amazing curative and therapeutic features that make them one of the most popular, safe, non-detrimental, and cytoprotective medicinal plant that can be used for prevention and treatment of many complicated diseases. Originally, N. sativa was used to treat migraines and allergy, and researches have shown its effectiveness in destroying cancer cells as well. The gastro protective effect of NS oil and its constituents has also been reported earlier; however, the complete perception on etiology and pathogenesis of gastric ulcer is not yet clear. Herein, we attempt to unveil some of the potential mechanisms exhibited by NS in preventing problems related to gastric ulcers. Gastric ailments like ulcers and tumors are the most common disorders of the gastro-intestinal tract in the present day life of the industrialized world. Gastric ulcer being a multifaceted problem exhibits complex etiology and is the fourth most common cause of cancer mortality. Drug interactions and toxicity are the main hindrances in chemotherapy. The existing merits and demerits of modern-day drugs make us turn toward the plant kingdom which may provide a valuable resource of novel potent natural compounds for pharmaceuticals or alternately, as dietary supplements. In this context, the revered phytotherapeutic N. sativa comes as a promising savior in today’s times. This review aims to summarize, both the functional and disease-related effects in the area of gastroenterology.
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Key Words
- 5-FU, 5-flourouracil
- COX, cyclooxygenase
- GI, gastrointestinal
- GSH, glutathione
- Gastric ulcer
- Gastro-protective
- Gastropathies
- LOX, lipoxygenase
- Medicinal plant
- NF-κB, nuclear transcription factor kappa B
- NS, Nigella sativa
- NSAIDs, non-steroidal anti-inflammatory drugs
- Nigella sativa
- PGs, prostaglandins
- PUFAs, polyunsaturated fatty acids
- ROS, reactive oxygen species
- TQ, thymoquinone
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Affiliation(s)
- Shahida A. Khan
- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Aziz M. Khan
- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Sajjad Karim
- Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Mohammad Amjad Kamal
- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Ghazi A. Damanhouri
- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Zeenat Mirza
- King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
- Corresponding author at: King Fahd Medical Research Center, King Abdulaziz University, Post Box No 80216, Jeddah 21589, Saudi Arabia. Tel.: +966 6401000x72074, mobile: +966 553017824; fax: +966 6952076.
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Bettenworth D, Lenz P, Krausewitz P, Brückner M, Ketelhut S, Domagk D, Kemper B. Quantitative stain-free and continuous multimodal monitoring of wound healing in vitro with digital holographic microscopy. PLoS One 2014; 9:e107317. [PMID: 25251440 PMCID: PMC4174518 DOI: 10.1371/journal.pone.0107317] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Accepted: 08/08/2014] [Indexed: 12/12/2022] Open
Abstract
Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.
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Affiliation(s)
- Dominik Bettenworth
- Department of Medicine B, University Hospital Münster, Münster, Germany
- * E-mail: (DB); (BK)
| | - Philipp Lenz
- Department of Medicine B, University Hospital Münster, Münster, Germany
| | | | - Markus Brückner
- Department of Medicine B, University Hospital Münster, Münster, Germany
| | - Steffi Ketelhut
- Center for Biomedical Optics and Photonics, University of Münster, Münster, Germany
- Biomedical Technology Center, University of Münster, Münster, Germany
| | - Dirk Domagk
- Department of Medicine B, University Hospital Münster, Münster, Germany
| | - Björn Kemper
- Center for Biomedical Optics and Photonics, University of Münster, Münster, Germany
- Biomedical Technology Center, University of Münster, Münster, Germany
- * E-mail: (DB); (BK)
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Chang RM, Wen LQ, Chang JX, Fu YR, Jiang ZP, Chen S. Repair of damaged intestinal mucosa in a mouse model of sepsis. World J Emerg Med 2014; 4:223-8. [PMID: 25215123 PMCID: PMC4129853 DOI: 10.5847/wjem.j.issn.1920-8642.2013.03.012] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2013] [Accepted: 06/12/2013] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND: The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injury in vivo in a mouse model of sepsis. METHODS: Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP. RESULTS: Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3–4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours. CONCLUSIONS: Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.
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Affiliation(s)
- Rui-Ming Chang
- Department of Emergency Medicine, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
| | - Li-Qiang Wen
- Department of Emergency Medicine, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
| | - Jian-Xing Chang
- Department of General Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
| | - Yu-Ru Fu
- Department of Emergency Medicine, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
| | - Zhi-Peng Jiang
- Department of Emergency Medicine, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
| | - Shuang Chen
- Department of General Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
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Modulation of intestinal epithelial cell proliferation, migration, and differentiation in vitro by Astragalus polysaccharides. PLoS One 2014; 9:e106674. [PMID: 25157577 PMCID: PMC4144960 DOI: 10.1371/journal.pone.0106674] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Accepted: 08/06/2014] [Indexed: 02/06/2023] Open
Abstract
Previous studies have shown that Astragalus polysaccharides (APS) can be used to treat general gastrointestinal disturbances including intestinal mucosal injury. However, the mechanism by which APS mediate this effect is unclear. In the present study, the effects of APS on proliferation, migration, and differentiation of intestinal epithelial cells (IEC-6) were assessed using an in vitro wounding model and colorimetric thiazolyl blue (MTT) assays. The effect of APS on IEC-6 cell differentiation was observed using a light microscope and scanning electron microscope, and the expression of differentiation-specific markers of IEC-6 cells, such as cytokeratin 18 (CK18), alkaline phosphatase (ALP), tight junction protein ZO-2, and sucrase-isomaltase (SI), was determined by immunofluorescence assay (IFA) and real-time PCR. In addition, APS-induced signaling pathways in IEC-6 cells were characterized. Our results indicated that APS significantly enhance migration and proliferation of IEC-6 cells in vitro. APS-treated IEC-6 cells have numerous microvilli on their apical surface and also highly express CK18, ALP, ZO-2, and SI. Moreover, APS-treated IEC-6 cells, in which the activity and expression level of ornithine decarboxylase (ODC) were significantly elevated, also exhibited an increase in cellular putrescine, whereas no significant increase in TGF-β levels was observed. These findings suggest that APS may enhance intestinal epithelial cell proliferation, migration, and differentiation in vitro by stimulating ODC gene expression and activity and putrescine production, independent of TGF-β. Exogenous administration of APS may provide a new approach for modulating intestinal epithelial wound restitution in vivo.
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Fiore APZP, Osaki LH, Gama P. Transforming growth factor β1 increases p27 levels via synthesis and degradation mechanisms in the hyperproliferative gastric epithelium in rats. PLoS One 2014; 9:e101965. [PMID: 25000203 PMCID: PMC4085006 DOI: 10.1371/journal.pone.0101965] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Accepted: 06/13/2014] [Indexed: 12/31/2022] Open
Abstract
Throughout postnatal development, the gastric epithelium expresses Transforming Growth Factor beta1 (TGFβ1), but it is also exposed to luminal peptides that are part of milk. During suckling period, fasting promotes the withdrawal of milk-born molecules while it stimulates gastric epithelial cell proliferation. Such response can be reversed by exogenous TGFβ1, as it directly affects cell cycle through the regulation of p27 levels. We used fasting condition to induce the hyperproliferation of gastric epithelial cells in 14-day-old Wistar rats, and evaluated the effects of TGFβ1 gavage on p27 expression, phosphorylation at threonine 187 (phospho-p27Thr187) and degradation. p27 protein level was reduced during fasting when compared to suckling counterparts, while phospho-p27Thr187/p27 ratio was increased. TGFβ1 gavage reversed this response, which was confirmed through immunostaining. By using a neutralizing antibody against TGFβ1, we found that it restored the p27 and phosphorylation levels detected during fasting, indicating the specific role of the growth factor. We noted that neither fasting nor TGFβ1 changed p27 expression, but after cycloheximide administration, we observed that protein synthesis was influenced by TGFβ1. Next, we evaluated the capacity of the gastric mucosa to degrade p27 and we recorded a higher concentration of the remaining protein in pups treated with TGFβ1, suggesting augmented stability under this condition. Thus, we showed for the first time that luminal TGFβ1 increased p27 levels in the rat gastric mucosa by up- regulating translation and reducing protein degradation. We concluded that such mechanisms might be used by rapidly proliferating cells to respond to milk-born TGFβ1 and food restriction.
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Affiliation(s)
- Ana P. Z. P. Fiore
- Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, São Paulo, SP Brazil
| | - Luciana H. Osaki
- Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, São Paulo, SP Brazil
| | - Patricia Gama
- Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, São Paulo, SP Brazil
- * E-mail:
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Seeliger C, Falldorf K, Sachtleben J, van Griensven M. Low-frequency pulsed electromagnetic fields significantly improve time of closure and proliferation of human tendon fibroblasts. Eur J Med Res 2014; 19:37. [PMID: 24996421 PMCID: PMC4096547 DOI: 10.1186/2047-783x-19-37] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2013] [Accepted: 06/19/2014] [Indexed: 12/14/2022] Open
Abstract
Background The promotion of the healing process following musculoskeletal injuries comprises growth factor signalling, migration, proliferation and apoptosis of cells. If these processes could be modulated, the healing of tendon tissue may be markedly enhanced. Here, we report the use of the Somagen™ device, which is certified for medical use according to European laws. It generates low-frequency pulsed electromagnetic fields that trigger effects of a nature that are yet to be determined. Methods A 1.5-cm wide, linear scrape was introduced into patellar tendon fibroblast cultures (N = 5 donors). Treatment was carried out every second day. The regimen was applied three times in total with 30 minutes comprising pulsed electromagnetic field packages with two fundamental frequencies (10 minutes of 33 Hz, 20 minutes of 7.8 Hz). Control cells remained untreated. All samples were analyzed for gap closure time, proliferation and apoptosis one week after induction of the scrape wound. Results The mean time for bridging the gap in the nontreated cells was 5.05 ± 0.33 days, and in treated cells, it took 3.35 ± 0.38 days (P <0.001). For cell cultures with scrape wounds, a mean value for BrdU incorporation of OD = 0.70 ± 0.16 was found. Whereas low-frequency pulsed electromagnetic fields treated samples showed OD = 1.58 ± 0.24 (P <0.001). However, the percentage of apoptotic cells did not differ between the two groups. Conclusions Our data demonstrate that low-frequency pulsed electromagnetic fields emitted by the Somagen™ device influences the in vitro wound healing of patellar tendon fibroblasts and, therefore, possibly increases wound healing potential.
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Affiliation(s)
| | | | | | - Martijn van Griensven
- Department of Trauma Surgery, Experimental Trauma Surgery, Klinikum rechts der Isar, Technical University Munich, Ismaninger Strasse 22, D-81675 Munich, Germany.
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Parlato M, Yeretssian G. NOD-like receptors in intestinal homeostasis and epithelial tissue repair. Int J Mol Sci 2014; 15:9594-627. [PMID: 24886810 PMCID: PMC4100112 DOI: 10.3390/ijms15069594] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Revised: 05/16/2014] [Accepted: 05/20/2014] [Indexed: 12/13/2022] Open
Abstract
The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.
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Affiliation(s)
- Marianna Parlato
- Department of Medicine, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
| | - Garabet Yeretssian
- Department of Medicine, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
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Jain SK, Baggerman EW, MohanKumar K, Namachivayam K, Jagadeeswaran R, Reyes VE, Maheshwari A. Amniotic fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2014; 306:G361-9. [PMID: 24407592 PMCID: PMC3949017 DOI: 10.1152/ajpgi.00272.2013] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.
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Affiliation(s)
- Sunil K. Jain
- 1Department of Pediatrics (Division of Neonatology), University of Texas Medical Branch, Galveston, Texas;
| | - Eric W. Baggerman
- 1Department of Pediatrics (Division of Neonatology), University of Texas Medical Branch, Galveston, Texas;
| | - Krishnan MohanKumar
- 2Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; ,3Center for Neonatal and Pediatric Gastrointestinal Disease, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois;
| | - Kopperuncholan Namachivayam
- 2Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; ,3Center for Neonatal and Pediatric Gastrointestinal Disease, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois;
| | - Ramasamy Jagadeeswaran
- 2Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; ,3Center for Neonatal and Pediatric Gastrointestinal Disease, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois;
| | - Victor E. Reyes
- 1Department of Pediatrics (Division of Neonatology), University of Texas Medical Branch, Galveston, Texas; ,4Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; and
| | - Akhil Maheshwari
- 2Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; ,3Center for Neonatal and Pediatric Gastrointestinal Disease, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois; ,5Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois
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Song HP, Li RL, Chen X, Wang YY, Cai JZ, Liu J, Chen WW. Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine--voltage-gated K+ channel pathway. JOURNAL OF ETHNOPHARMACOLOGY 2014; 152:163-172. [PMID: 24417867 DOI: 10.1016/j.jep.2013.12.049] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/15/2013] [Revised: 10/30/2013] [Accepted: 12/30/2013] [Indexed: 06/03/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Atractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model. MATERIALS AND METHODS A wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5 μmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5 mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200 μg/mL), DFMO plus SPD and DFMO plus AMK for 24h. The membrane potential (MP) and cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. RESULTS (1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca(2+)]cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca(2+)]cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells. CONCLUSIONS The results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.
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Affiliation(s)
- Hou-Pan Song
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China
| | - Ru-Liu Li
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China.
| | - Xu Chen
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China
| | - Yi-Yu Wang
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China
| | - Jia-Zhong Cai
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China
| | - Jia Liu
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China
| | - Wei-Wen Chen
- Spleen and Stomach Institute, Guangzhou University of Chinese Medicine, 12 Airport Road, Baiyun District, Guangzhou 510405, PR China; Research Center of Medicinal Plant Resource Science and Engineering, Guangzhou University of Chinese Medicine, 232 WaiHuan East Road, Guangzhou University Town, Guangzhou 510006, PR China
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Yamanaka O, Yuan Y, Coulson-Thomas VJ, Gesteira TF, Call MK, Zhang Y, Zhang J, Chang SH, Xie C, Liu CY, Saika S, Jester JV, Kao WWY. Lumican binds ALK5 to promote epithelium wound healing. PLoS One 2013; 8:e82730. [PMID: 24367547 PMCID: PMC3867403 DOI: 10.1371/journal.pone.0082730] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2013] [Accepted: 11/06/2013] [Indexed: 12/30/2022] Open
Abstract
Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.
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Affiliation(s)
- Osamu Yamanaka
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Yong Yuan
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | | | - Tarsis Ferreira Gesteira
- Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio, United States of America
| | - Mindy K. Call
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Yujin Zhang
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Jianhua Zhang
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Shao-Hsuan Chang
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Changchun Xie
- Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Chia-Yang Liu
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Shizuya Saika
- Department of Ophthalmology, Wakayama Medical College, 811-1 Kimiidera, Wakayama, Japan
| | - James V. Jester
- Gavin Herbert Eye Institute, Ophthalmology, University of California Irvine, Irvine, California, United States of America
| | - Winston W-Y Kao
- Deparment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, United States of America
- * E-mail:
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The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut. Cytokine Growth Factor Rev 2013; 25:45-55. [PMID: 24332927 DOI: 10.1016/j.cytogfr.2013.11.001] [Citation(s) in RCA: 75] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2013] [Accepted: 11/19/2013] [Indexed: 02/07/2023]
Abstract
Transforming growth factor (TGF)-β, a pleiotropic cytokine released by both immune and non-immune cells in the gut, exerts an important tolerogenic action by promoting regulatory T cell differentiation. TGF-β also enhances enterocyte migration and regulates extracellular matrix turnover, thereby playing a crucial role in tissue remodeling in the gut. In this review we describe the mechanisms by which abnormal TGF-β signaling impairs intestinal immune tolerance and tissue repair, thus predisposing to the onset of immune-mediated bowel disorders, such as inflammatory bowel disease and celiac disease. Additionally, we will discuss potential therapeutic strategies aiming at restoring physiologic TGF-β signaling in chronic intestinal diseases.
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TGF-β affects enterocyte turnover in correlation with TGF-β receptor expression after massive small bowel resection. J Pediatr Gastroenterol Nutr 2012; 55:721-7. [PMID: 22711000 DOI: 10.1097/mpg.0b013e318263ec18] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
OBJECTIVES In the present study, we evaluated the effect of transforming growth factor-beta 2 (TGF-β2)-enriched diet on enterocyte turnover and correlated it with TGF-β2 receptor expression along the villus-crypt axis in a rat model of short bowel syndrome (SBS). METHODS CaCo-2 cells were incubated with increasing concentrations of TGF-β2. Alamar Blue reduction test was used for investigation of cell viability and evaluation of cell apoptosis was assessed by flow cytometry. Male rats were divided into 4 groups: Sham rats underwent bowel transection, Sham TGF-β rats were treated with diet enriched with TGF-β2, SBS rats underwent a 75% bowel resection, and SBS TGF-β rats were fed a diet enriched with TGF-β2 after bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at sacrifice. TGF-β2r expression in villus tips, lateral villi and crypts was assessed by immunohistochemistry. The effect of TGF-β2 on enterocyte turnover for each compartment was evaluated in correlation with TGF-β2r expression. RESULTS Incubation of CaCo-2 cells with TGF-β2 resulted in a significant decrease in cell viability and increased cell apoptosis. TGF-β2r expression in crypts increased in SBS rats (vs sham) and was accompanied by decreased cell proliferation and increased cell apoptosis following TGF-β2 administration. A significant decrease in TGF-β2r expression at villous tips in SBS rats was accompanied by a decreased cell apoptosis in this compartment following exposure to TGF-β2-enriched diet. CONCLUSIONS In a rat model of SBS, the inhibiting effect of TGF-β2 on enterocyte turnover correlates with TGF-β2 receptor expression along the villus-crypt axis.
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Hindriksen S, Bijlsma MF. Cancer Stem Cells, EMT, and Developmental Pathway Activation in Pancreatic Tumors. Cancers (Basel) 2012; 4:989-1035. [PMID: 24213498 PMCID: PMC3712732 DOI: 10.3390/cancers4040989] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2012] [Revised: 10/02/2012] [Accepted: 10/09/2012] [Indexed: 12/15/2022] Open
Abstract
Pancreatic cancer is a disease with remarkably poor patient survival rates. The frequent presence of metastases and profound chemoresistance pose a severe problem for the treatment of these tumors. Moreover, cross-talk between the tumor and the local micro-environment contributes to tumorigenicity, metastasis and chemoresistance. Compared to bulk tumor cells, cancer stem cells (CSC) have reduced sensitivity to chemotherapy. CSC are tumor cells with stem-like features that possess the ability to self-renew, but can also give rise to more differentiated progeny. CSC can be identified based on increased in vitro spheroid- or colony formation, enhanced in vivo tumor initiating potential, or expression of cell surface markers. Since CSC are thought to be required for the maintenance of a tumor cell population, these cells could possibly serve as a therapeutic target. There appears to be a causal relationship between CSC and epithelial-to-mesenchymal transition (EMT) in pancreatic tumors. The occurrence of EMT in pancreatic cancer cells is often accompanied by re-activation of developmental pathways, such as the Hedgehog, WNT, NOTCH, and Nodal/Activin pathways. Therapeutics based on CSC markers, EMT, developmental pathways, or tumor micro-environment could potentially be used to target pancreatic CSC. This may lead to a reduction of tumor growth, metastatic events, and chemoresistance in pancreatic cancer.
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Affiliation(s)
- Sanne Hindriksen
- Laboratory for Experimental Oncology and Radiobiology, Academic Medical Centre, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands.
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26
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Zhao QJ, Yu YB, Zuo XL, Dong YY, Li YQ. Milk fat globule-epidermal growth factor 8 is decreased in intestinal epithelium of ulcerative colitis patients and thereby causes increased apoptosis and impaired wound healing. Mol Med 2012; 18:497-506. [PMID: 22204000 DOI: 10.2119/molmed.2011.00369] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2011] [Accepted: 12/20/2011] [Indexed: 12/19/2022] Open
Abstract
Milk fat globule-epidermal growth factor 8 (MFG-E8) plays an important role in maintaining intestinal barrier homeostasis and accelerating intestinal restitution. However, studies of MFG-E8 expression in humans with ulcerative colitis are lacking. We examined MFG-E8 expression in colonic mucosal biopsies from ulcerative colitis patients and healthy controls (n = 26 each) by real-time quantitative polymerase chain reaction (PCR), Western blot analysis and immunohistochemistry. MFG-E8 mRNA and protein expression was lower in ulcerative colitis patients than in controls. MFG-E8 expression was inversely correlated with mucosal inflammatory activity and clinical disease activity in patients. MFG-E8 was present in human intestinal epithelial cells both in vivo and in vitro. Apoptosis induction was also detected in the intestinal epithelium of ulcerative colitis patients by terminal-deoxynucleoitidyl transferase mediated nick-end labeling assay. We used lentiviral vectors encoding human MFG-E8 targeting short hairpin RNA to obtain MFG-E8 knockdown intestinal epithelia cell clones. MFG-E8 knockdown could promote apoptosis in intestinal epithelial cell lines, accompanied by a decrease in level of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) and induction of the proapoptotic protein BCL2-associated protein X (BAX). The addition of recombinant human MFG-E8 led to decreased BAX and cleaved caspase-3 levels and induction of BCL-2 level in intestinal epithelia cells. MFG-E8 knockdown also attenuated wound healing on scratch assay of intestinal epithelial cells. The mRNA level of intestinal trefoid factor 3, a pivotal factor in intestinal epithelial cell migration and restitution, was downregulated with MFG-E8 knockdown. In conclusion, we demonstrated that decreased colonic MFG-E8 expression in patients with ulcerative colitis may be associated with mucosal inflammatory activity and clinical disease activity through basal cell apoptosis and preventing tissue healing in the pathogenesis of ulcerative colitis.
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Affiliation(s)
- Qiu-jie Zhao
- Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, Shandong, China
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Hwang S, Zimmerman NP, Agle KA, Turner JR, Kumar SN, Dwinell MB. E-cadherin is critical for collective sheet migration and is regulated by the chemokine CXCL12 protein during restitution. J Biol Chem 2012; 287:22227-40. [PMID: 22549778 DOI: 10.1074/jbc.m112.367979] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Chemokines and other immune mediators enhance epithelial barrier repair. The intestinal barrier is established by highly regulated cell-cell contacts between epithelial cells. The goal of these studies was to define the role for the chemokine CXCL12 in regulating E-cadherin during collective sheet migration during epithelial restitution. Mechanisms regulating E-cadherin were investigated using Caco2(BBE) and IEC-6 model epithelia. Genetic knockdown confirmed a critical role for E-cadherin in in vitro restitution and in vivo wound repair. During restitution, both CXCL12 and TGF-β1 tightened the monolayer by decreasing the paracellular space between migrating epithelial cells. However, CXCL12 differed from TGF-β1 by stimulating the significant increase in E-cadherin membrane localization during restitution. Chemokine-stimulated relocalization of E-cadherin was paralleled by an increase in barrier integrity of polarized epithelium during restitution. CXCL12 activation of its cognate receptor CXCR4 stimulated E-cadherin localization and monolayer tightening through Rho-associated protein kinase activation and F-actin reorganization. These data demonstrate a key role for E-cadherin in intestinal epithelial restitution.
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Affiliation(s)
- Soonyean Hwang
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
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Rieder F, Karrasch T, Ben-Horin S, Schirbel A, Ehehalt R, Wehkamp J, de Haar C, Velin D, Latella G, Scaldaferri F, Rogler G, Higgins P, Sans M. Results of the 2nd scientific workshop of the ECCO (III): basic mechanisms of intestinal healing. J Crohns Colitis 2012; 6:373-85. [PMID: 22405177 DOI: 10.1016/j.crohns.2011.11.009] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/12/2011] [Accepted: 11/13/2011] [Indexed: 02/08/2023]
Abstract
The second scientific workshop of the European Crohn's and Colitis Organization (ECCO) focused on the relevance of intestinal healing for the disease course of inflammatory bowel disease (IBD). The objective was to better understand basic mechanisms, markers for disease prediction, detection and monitoring of intestinal healing, impact of intestinal healing on the disease course of IBD as well as therapeutic strategies. The results of this workshop are presented in four separate manuscripts. This section describes basic mechanisms of intestinal healing, identifies open questions in the field and provides a framework for future studies.
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Affiliation(s)
- Florian Rieder
- Department of Gastroenterology & Hepatology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, USA.
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Chailler P, Beaulieu JF, Ménard D. Isolation and functional studies of human fetal gastric epithelium in primary culture. Methods Mol Biol 2012; 806:137-55. [PMID: 22057450 DOI: 10.1007/978-1-61779-367-7_10] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistence of viable chief cells able to synthesize and secrete gastric digestive enzymes, i.e., pepsinogen and gastric lipase. In wounding experiments, epithelial restitution occurred in serum-reduced conditions and was modulated by exogenous agents. This culture system is thus representative of the foveolus-gland axis and offers new perspectives to establish the influence of individual growth factors and extracellular matrix components as well as their combinatory effects on gastric epithelium homeostasis.
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Affiliation(s)
- Pierre Chailler
- CIHR Team on Digestive Epithelium, Département d'anatomie et de biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada
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Agle KA, Vongsa RA, Dwinell MB. Chemokine stimulation promotes enterocyte migration through laminin-specific integrins. Am J Physiol Gastrointest Liver Physiol 2011; 301:G968-80. [PMID: 21921288 PMCID: PMC3233784 DOI: 10.1152/ajpgi.00208.2011] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Intestinal homeostasis is regulated in part by the single cell layer of the mucosal epithelium. This physical barrier is a prominent part of the innate immune system and possesses an intrinsic ability to heal damage and limit infection. The restitutive epithelial migration phase of healing requires dynamic integrin adhesion to the extracellular matrix. Previously, we have shown that the homeostatic chemokine CXCL12 utilizes intracellular calcium to increase enterocyte migration on laminin. The aim of these studies was to investigate integrin specificity and, in turn, functional responses elicited by CXCL12 stimulation. Analysis of cellular adhesion and spreading revealed CXCL12 preferentially activated laminin-specific integrins compared with collagen IV-binding integrins. Laminin-specific cell adhesion and spreading elicited by CXCL12 was dependent on intracellular calcium. CXCL12 increased activated β1-integrins on the surface of epithelial cells compared with untreated cells. RT-PCR confirmed expression of the laminin-binding integrins-α3β1, -α6β1, and -α6β4. Interestingly, shRNA-mediated depletion of laminin-specific α3- or α6-integrin subunits revealed differential functions. α3-Integrin knockdown reduced basal as well as inducible restitution. Depletion of α6-integrin specifically abolished CXCL12-stimulated, but not TGF-β1 or basal, migration. Depletion with either shα3-integrin or shα6-integrin prevented CXCL12-evoked cell spreading. Our data indicate that CXCL12 stimulates the inside-out activation of laminin-specific integrins to promote cell migratory functions. Together, our findings support the notion that extracellular mediators within the gastrointestinal mucosa coordinate cell-matrix interactions during epithelial restitution.
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Affiliation(s)
- Kimberle A. Agle
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Rebecca A. Vongsa
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Michael B. Dwinell
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin
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Karrasch T, Spaeth T, Allard B, Jobin C. PI3K-dependent GSK3ß(Ser9)-phosphorylation is implicated in the intestinal epithelial cell wound-healing response. PLoS One 2011; 6:e26340. [PMID: 22039465 PMCID: PMC3198390 DOI: 10.1371/journal.pone.0026340] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2011] [Accepted: 09/25/2011] [Indexed: 01/12/2023] Open
Abstract
Introduction The ability of the intestinal epithelial barrier to respond to various injurious insults is an essential component of intestinal homeostasis. However, the molecular mechanisms responsible for wound-healing and repair in the intestine are poorly understood. The glycogen synthase kinase 3ß (GSK3ß) has been implicated in various biological processes such as cellular motility, cell spreading and recently inflammation. Aim To investigate the role of GSK3ß in intestinal epithelial cell restitution. Methods Rat intestinal epithelial IEC18 cells were serum-starved for 16 to 24h and wounded by multiple scraping. Akt(Ser473)-, GSK3ß(Ser9)- and RelA(Ser536)-phosphorylation were determined by Western blot using specific phospho-antibodies. The inhibitors AG1478 (1 µM) and Ly294002 (25 µM) were used to block EGF-R autophosphorylation and PI3K-activation, respectively. ß-catenin/LEF/TCF dependent transcription was determined by reporter gene assay (TOP/FOP system). C-myc gene expression was evaluated by real-time RT-PCR. GSK3ß−/− mouse embryonic fibroblasts were used to characterize the role of GSK3ß in wounding-induced cell migration. Results Wounding induced GSK3ß(Ser9) phosphorylation in IEC-18 cells, which led to ß-catenin accumulation as well as nuclear translocation of ß-catenin. ß-catenin stabilization/nuclear translocation led to enhanced LEF-TCF transcriptional activity and subsequent c-myc mRNA accumulation in wounded cell monolayers. Blocking PI3K/Akt signaling with Ly294002 prevented wound-induced GSK3ß(Ser9) phosphorylation as well as ß-catenin nuclear translocation and significantly attenuated restitution. Additionally, wounding induced rapid NF-kB(Ser536) phosphorylation, which was inhibited by AG1478, but not by Ly294002. GSK3ß−/− cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells. Conclusion We conclude that PI3K-mediated GSK3ß phosphorylation is involved in the intestinal epithelial wound-healing response. Phosphorylation of GSK3ß may be important for intestinal restitution by promoting cell motility in response to wounding.
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Affiliation(s)
- Thomas Karrasch
- Department of Internal Medicine I, University of Regensburg, Regensburg, Germany.
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Sun LX, Chen LH, Lin ZB, Qin Y, Zhang JQ, Yang J, Ma J, Ye T, Li WD. Effects of Ganoderma lucidum polysaccharides on IEC-6 cell proliferation, migration and morphology of differentiation benefiting intestinal epithelium healing in vitro. J Pharm Pharmacol 2011; 63:1595-603. [PMID: 22060291 DOI: 10.1111/j.2042-7158.2011.01367.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
OBJECTIVES Restoration of epithelial continuity in the intestinal surface after extensive destruction is important since intestinal epithelial cells stand as a boundary between the body's internal and external environment. Polysaccharides from Ganoderma lucidum (Gl-PS) may benefit intestinal epithelial wound healing in different aspects, which awaits clarification. To identify potential effects, a non-transformed small-intestinal epithelial cell line, IEC-6 cells, was used. METHODS Effects on epithelial cell proliferation, migration, morphology of differentiation and transforming growth factor beta (TGF-β) protein expression, as well as the cellular ornithine decarboxylase (ODC) mRNA and c-Myc mRNA expression, were assessed, respectively, by MTT assay, wound model in vitro, observation under a microscope after hematoxylin and eosin staining, enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction assays. KEY FINDINGS It was shown that Gl-PS stimulated IEC-6 cell proliferation and migration significantly in a dose-dependent manner; 10 µg/ml Gl-PS improved the morphology of differentiation in IEC-6 cells. Inefficacy in expression of TGF-β in IEC-6 cells indicated a possible TGF-β independent action of Gl-PS. However, Gl-PS increased ODC mRNA and c-Myc mRNA expression in a dose-dependent manner, indicating, at least partially possible involvement of ODC and c-Myc gene expression in improvement of intestinal wound healing. CONCLUSIONS These results suggest the potential usefulness of Gl-PS to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.
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Affiliation(s)
- Li-Xin Sun
- Department of Pharmacology, Basic Medical School, Peking University Health Science Center, Beijing Affiliated Hospital of Chengde Medical College, Chengde, Hebei Province, China
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Hamady ZZR, Scott N, Farrar MD, Wadhwa M, Dilger P, Whitehead TR, Thorpe R, Holland KT, Lodge JPA, Carding SR. Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-β1 under the control of dietary xylan 1. Inflamm Bowel Dis 2011; 17:1925-35. [PMID: 21830271 DOI: 10.1002/ibd.21565] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/04/2010] [Accepted: 10/11/2010] [Indexed: 12/12/2022]
Abstract
BACKGROUND While cytokine therapy and the use of immunosuppressive cytokines such as transforming growth factor-β (TGF-β) offer great potential for the treatment of inflammatory bowel disease (IBD), issues concerning formulation, stability in vivo, delivery to target tissues, and potential toxicity need to be addressed. In consideration of these problems we engineered the human commensal bacterium Bacteroides ovatus for the controlled in situ delivery of TGF-β(1) and treatment of colitis. METHODS Sequence encoding the human tgf-β1 gene was cloned downstream of the xylanase promoter in the xylan operon of B. ovatus by homologous recombination. Resulting recombinants (BO-TGF) were tested for TGF-β production in the presence and absence of polysaccharide xylan in vitro and in vivo, and used to treat experimental murine colitis. Clinical and pathological scores were used to assess the effectiveness of therapy. Colonic inflammatory markers including inflammatory cytokine expression were assessed by colorimetric assay and real-time polymerase chain reaction (PCR). RESULTS BO-TGF secreted high levels of biologically active dimeric TGF-β in vitro and in vivo in a xylan-controlled manner. Administration of xylan in drinking water to BO-TGF-treated mice resulted in a significant clinical improvement of colitis, accelerating healing of damaged colonic epithelium, reducing inflammatory cell infiltration, reducing expression of proinflammatory cytokines, and promoting production of mucin-rich goblet cells in colonic crypts. These beneficial effects are comparable and in most cases superior to that achieved by conventional steroid therapy. CONCLUSIONS This novel drug delivery system has potential for the targeted and controlled delivery of TGF-β(1) and other immunotherapeutic agents for the long-term management of various bowel disorders.
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Affiliation(s)
- Zaed Z R Hamady
- Institute of Molecular and Cellular Biology, University of Leeds, Leeds, UK
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Goldsmith JR, Uronis JM, Jobin C. Mu opioid signaling protects against acute murine intestinal injury in a manner involving Stat3 signaling. THE AMERICAN JOURNAL OF PATHOLOGY 2011; 179:673-83. [PMID: 21801866 DOI: 10.1016/j.ajpath.2011.04.032] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/17/2010] [Revised: 03/04/2011] [Accepted: 04/05/2011] [Indexed: 12/16/2022]
Abstract
Opiates have long been used as analgesics to relieve pain associated with various medical conditions. Here, we evaluated the effect and mechanism of mu opioid signaling on the intestinal wound healing response and assessed downstream pathways known to be protective against intestinal injury. Mice (C57BL/6) were exposed to 3% dextran sodium sulfate (DSS) for 7 days or 4% DSS for 5 days followed by 7 days of water. The mu opioid receptor (MOR)-specific agonist [D-Arg2,Lys4]dermorphin-(1,4)-amide (DALDA) and the antagonist cyprodime were injected s.c. daily for in vivo studies or used for in vitro analysis. We found that MOR activation attenuated DSS-induced histologic and gross intestinal injury and weight loss; diminished Ifng, Tnf, and Il6 mRNA expression; and promoted intestinal healing during recovery. DALDA also enhanced colonocyte proliferation (Ki-67 staining) by 350%. MOR activation increased Stat3 phosphorylation in both DALDA-treated mice and the CMT-93 cell line. Importantly, DALDA-induced colonocyte migration was completely ablated by shStat3 knockdown. Together, this work shows that MOR activation protects against and enhances recovery from DSS-induced intestinal injury. This is associated with an increase in Stat3 activation. Furthermore, Stat3 is required for DALDA-induced colonocyte migration. Consequently, manipulation of MOR signaling may represent a novel means to promote mucosal healing and to maintain intestinal homeostasis after intestinal injury.
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Affiliation(s)
- Jason R Goldsmith
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA
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Iizuka M, Konno S. Wound healing of intestinal epithelial cells. World J Gastroenterol 2011; 17:2161-71. [PMID: 21633524 PMCID: PMC3092866 DOI: 10.3748/wjg.v17.i17.2161] [Citation(s) in RCA: 189] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2010] [Revised: 01/15/2011] [Accepted: 01/22/2011] [Indexed: 02/06/2023] Open
Abstract
The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing.
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Hoffmann P, Sturm A, Stein J, Dignass AU. Interferon-γ modulates intestinal epithelial cell function in-vitro through a TGFβ-dependent mechanism. ACTA ACUST UNITED AC 2011; 168:27-31. [PMID: 21385594 DOI: 10.1016/j.regpep.2011.02.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2010] [Revised: 02/02/2011] [Accepted: 02/18/2011] [Indexed: 10/18/2022]
Abstract
INTRODUCTION Interferon γ (IFNγ) has been originally identified by its anti-viral activity and has been demonstrated to act as potent modulator of the immune system with a range of target cells limited largely to immune cell populations. Although IFNγ has been shown to directly affect the barrier function of intestinal epithelial cells, only limited information is available about other functional effects of IFNγ on intestinal epithelial cells. METHODS The effects on intestinal epithelial cell migration were studied using a previously described in-vitro model of epithelial restitution in confluent IEC-6 cell monolayers. Intestinal epithelial cell proliferation rates were assessed in various human and rat intestinal and colon epithelial cell lines using colorimetric MTT assays. Apoptosis of IEC-6 cells exposed to IFNγ was assessed by flow cytometry. In addition, transforming growth factor β mRNA expression after IFNγ treatment of IEC-6 cells was assessed by Northern blot analysis. RESULTS IFNγ significantly stimulated intestinal epithelial cell migration in an in-vitro wounding model. Furthermore, IFNγ caused a significant dose-dependent inhibition of epithelial cell proliferation in non-transformed small intestinal IEC-6 cells and human colon cancer-derived HT-29 cells and no significant rates of apoptosis were detected in the exposed epithelial cells. The effect of IFNγ on epithelial cell migration and proliferation could be completely blocked by neutralizing antibodies against TGFβ indicating that these effects are mediated through a TGFβ dependent pathway. In addition, increased expression of TGFβ1 mRNA by IEC-6 cells after treatment with IFNγ supports the hypothesis that IFNγ modulates intestinal epithelial cell function through a TGFβ-dependent pathway. CONCLUSION These studies suggest that IFNγ produced by constituents of the mucosal immune system modulates epithelial cell functions with relevance for intestinal wound healing and may play a role in preserving the integrity of the intestinal epithelium following various forms of injuries.
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Affiliation(s)
- Peter Hoffmann
- Department of Internal Medicine I, Kliniken Essen Mitte, Germany
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Dias A, Garcia C, Majewski M, Wallner G, McCallum RW, Poplawski C, Sarosiek J. Gastric juice prostaglandins and peptide growth factors as potential markers of chronic atrophic gastritis, intestinal metaplasia and gastric cancer: their potential clinical implications based on this pilot study. Dig Dis Sci 2011; 56:3220-5. [PMID: 21695403 PMCID: PMC3208809 DOI: 10.1007/s10620-011-1758-z] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2010] [Accepted: 05/14/2011] [Indexed: 12/23/2022]
Abstract
BACKGROUND Gastric secretion can provide valuable information especially when Helicobacter pylori (Hp) infection results in chronic atrophic gastritis (CAG) and intestinal metaplasia (IM) preceding adenocarcinoma (AdCa). AIMS Looking for a potential biomarker of malignant transformation in the setting of chronic inflammation we studied the levels of prostaglandin E2 (PGE(2)), as well as peptide growth factors [epidermal growth factor (EGF) and transforming growth factor α (TGFα)], harbingers of injury and repair, in gastric juice aspirated at endoscopy from patients with CAG, CAG/IM, AdCa, and controls. METHODS The PGE(2), EGF and TGFα concentrations in the gastric juice were measured using radioimmunoassays (RIAs). RESULTS In patients with AdCa gastric juice PGE(2) increased fivefold versus controls (P < 0.01) and almost threefold versus patients with CAG (P < 0.05). The EGF levels in patients with AdCa were fourfold higher versus controls (P < 0.001) and almost threefold higher versus CAG (P < 0.05). In patients with CAG/IM the EGF levels were also almost 3 times higher versus controls. The TGFα levels in patients with AdCa were half the value of controls and CAG (P < 0.05). In patients with CAG/IM the levels were as low as 1/5 of controls or CAG (P < 0.05). CONCLUSIONS Testing the gastric juice for PGE(2), EGF, and TGFα in patients with endoscopy and biopsy proven CAG, may be helpful in follow up of patients who may potentially progress to IM and ultimately AdCa. This could be considered as an adjunct to histologic assessment especially that even the best surveillance biopsy specimen regimens are inherited with sampling errors.
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Affiliation(s)
- Ajoy Dias
- Department of Internal Medicine, TTUHSC Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, TX 79905-2709 USA
| | - Cesar Garcia
- Department of Internal Medicine, TTUHSC Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, TX 79905-2709 USA
| | | | | | - Richard W. McCallum
- Department of Internal Medicine, TTUHSC Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, TX 79905-2709 USA
| | | | - Jerzy Sarosiek
- Department of Internal Medicine, TTUHSC Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, 4800 Alberta Avenue, El Paso, TX 79905-2709 USA
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Wendt MK, Schiemann WP. Therapeutic targeting of the focal adhesion complex prevents oncogenic TGF-beta signaling and metastasis. Breast Cancer Res 2010; 11:R68. [PMID: 19740433 PMCID: PMC2790843 DOI: 10.1186/bcr2360] [Citation(s) in RCA: 129] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2009] [Revised: 09/01/2009] [Accepted: 09/09/2009] [Indexed: 02/07/2023] Open
Abstract
Introduction Mammary tumorigenesis is associated with the increased expression of several proteins in the focal adhesion complex, including focal adhesion kinase (FAK) and various integrins. Aberrant expression of these molecules occurs concomitant with the conversion of TGF-β function from a tumor suppressor to a tumor promoter. We previously showed that interaction between β3 integrin and TβR-II facilitates TGF-β-mediated oncogenic signaling, epithelial-mesenchymal transition (EMT), and metastasis. However, the molecular mechanisms by which the focal adhesion complex contributes to β3 integrin:TβR-II signaling and the oncogenic conversion of TGF-β remain poorly understood. Methods FAK expression and activity were inhibited in normal and malignant mammary epithelial cells (MECs) either genetically by using lentiviral-mediated delivery of shRNAs against FAK, or pharmacologically through in vitro and in vivo use of the FAK inhibitors, PF-562271 and PF-573228. Altered Smad2/3 and p38 MAPK activation, migration, EMT, and invasion in response to TGF-β1 were monitored in FAK-manipulated cells. TβR-II expression was increased in metastatic breast cancer cells by retroviral transduction, and the metastasis of FAK- and TβR-II-manipulated tumors was monitored by using bioluminescent imaging. Results TGF-β stimulation of MECs stabilized and activated FAK in a β3 integrin- and Src-dependent manner. Furthermore, by using the human MCF10A breast cancer progression model, we showed that increased FAK expression in metastatic breast cancer cells mirrored the acquisition of enhanced activation of p38 MAPK by TGF-β. Administering FAK inhibitors or rendering metastatic breast cancer cells FAK deficient abrogated the interaction between β3 integrin and TβR-II, thereby preventing TGF-β from (a) activating p38 MAPK; (b) stimulating MEC invasion, migration, and EMT; and (c) inducing early primary tumor dissemination to the lungs. Finally, in contrast to FAK depletion, adjuvant FAK chemotherapy of mammary tumors decreased their growth in part by diminished macrophage tumor infiltration. Conclusions Our studies identify an essential function for FAK in mediating the interaction between β3 integrin and TβR-II, and thus in facilitating the oncogenic conversion of TGF-β required for mammary tumor metastasis. Furthermore, this study establishes chemotherapeutic targeting of FAK as an effective, two-pronged approach in preventing tumor progression both by decreasing innate immune cell infiltration, and by inhibiting early TGF-β-dependent metastasis.
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Affiliation(s)
- Michael K Wendt
- Department of Pharmacology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA.
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Bombesin enhances TGF-beta growth inhibitory effect through apoptosis induction in intestinal epithelial cells. ACTA ACUST UNITED AC 2009; 158:26-31. [PMID: 19631696 DOI: 10.1016/j.regpep.2009.07.010] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2009] [Revised: 06/01/2009] [Accepted: 07/02/2009] [Indexed: 12/17/2022]
Abstract
Mammalian intestinal epithelium undergoes continuous cell turn over, with cell proliferation in the crypts and apoptosis in the villus. Both transforming growth factor (TGF)-beta and gastrin-releasing peptide (GRP) are involved in the regulation of intestinal epithelial cells for division, differentiation, adhesion, migration and death. Previously, we have shown that TGF-beta and bombesin (BBS) synergistically induce cyclooxygenase-2 (COX-2) expression and subsequent prostaglandin E(2) (PGE2) production through p38(MAPK) in rat intestinal epithelial cell line stably transfected with GRP receptor (RIE/GRPR), suggesting the interaction between TGF-beta signaling pathway and GRPR. The current study examined the biological responses of RIE/GRPR cells to TGF-beta and BBS. Treatment with TGF-beta1 (40 pM) and BBS (100 nM) together synergistically inhibited RIE/GRPR growth and induced apoptosis. Pretreatment with SB203580 (10 microM), a specific inhibitor of p38(MAPK), partially blocked the synergistic effect of TGF-beta and BBS on apoptosis. In conclusion, BBS enhanced TGF-beta growth inhibitory effect through apoptosis induction, which is at least partially mediated by p38(MAPK).
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40
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Ey B, Eyking A, Gerken G, Podolsky DK, Cario E. TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury. J Biol Chem 2009; 284:22332-22343. [PMID: 19528242 DOI: 10.1074/jbc.m901619200] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.
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Affiliation(s)
- Birgit Ey
- Division of Gastroenterology and Hepatology, University Hospital of Essen, and Medical School, University of Duisburg-Essen, 45147 Essen, Germany
| | - Annette Eyking
- Division of Gastroenterology and Hepatology, University Hospital of Essen, and Medical School, University of Duisburg-Essen, 45147 Essen, Germany
| | - Guido Gerken
- Division of Gastroenterology and Hepatology, University Hospital of Essen, and Medical School, University of Duisburg-Essen, 45147 Essen, Germany
| | - Daniel K Podolsky
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390
| | - Elke Cario
- Division of Gastroenterology and Hepatology, University Hospital of Essen, and Medical School, University of Duisburg-Essen, 45147 Essen, Germany
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Ogias D, de Andrade Sá ER, Alvares EP, Gama P. Opposite effects of fasting on TGF-beta3 and TbetaRI distribution in the gastric mucosa of suckling and early weanling rats. Nutrition 2009; 26:224-9. [PMID: 19524404 DOI: 10.1016/j.nut.2009.03.014] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2008] [Revised: 03/09/2009] [Accepted: 03/27/2009] [Indexed: 12/11/2022]
Abstract
OBJECTIVE Our aim was to evaluate the effects of a dietary regimen (suckling or early weaning) and feeding status (fed or fasted) on the distribution of transforming growth factor-beta3 (TGF-beta3) and TGF receptor-I (TbetaRI) in the gastric epithelium of pups. METHODS Wistar rats were used. At 15 d, half of the pups were separated from dams and fed with hydrated powered chow. On day 17, suckling and early weanling rats were subjected to fasting (17h). Four different conditions were established: suckling fed and fasted and early weanling fed and fasted. At 18 d stomachs were collected under anesthesia and were fixed in 4% formaldehyde for immunohistochemistry. The number of immunostained epithelial cells per microscopic field was determined for TGF-beta3 and TbetaRI in longitudinal sections from the gastric mucosa. RESULTS We found that during suckling, fasting reduced the number of immunolabeled cells per field of both molecules when compared with the fed group (P<0.05), whereas in early weaning, food restriction increased TGF-beta3 and TbetaRI distributions (P<0.05). We also observed that TGF-beta3 and TbetaRI were more concentrated in parietal cells in the upper gland in suckling pups, whereas after early weaning these were displaced to parietal and chief cells at the bottom of the gland. CONCLUSION Suckling and early weaning directly influence TGF-beta3 and TbetaRI distributions in the gastric epithelium in response to fasting, such that early weaning anticipates the effects observed in adult rats. Furthermore, the differential concentrations of TGF-beta3 and TbetaRI indicate that they might be important for cell proliferation events in growth control.
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Affiliation(s)
- Daniela Ogias
- Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
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Inoue S, Nakase H, Matsuura M, Mikami S, Ueno S, Uza N, Chiba T. The effect of proteasome inhibitor MG132 on experimental inflammatory bowel disease. Clin Exp Immunol 2009; 156:172-82. [PMID: 19220323 DOI: 10.1111/j.1365-2249.2008.03872.x] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Immunoproteasome up-regulation enhances the processing of nuclear factor-kappaB (NF-kappaB) and degradation of IkappaBalpha, which correlates with increased amounts of NF-kappaB in the various cells. Aberrant activation of NF-kappaB is involved in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to elucidate the effect of proteasome inhibitor MG132 on experimental IBD. We investigated the effects of MG132 on intestinal inflammation and epithelial regeneration in both interleukin-10-deficient (IL-10(-/-)) mice and mice with dextran sulphate sodium (DSS)-induced colitis. Body weight, histological findings and tumour necrosis factor (TNF)-alpha mRNA expression, epithelial cell proliferation and NF-kappaB p65 activity in colonic tissues were examined. The effects of MG132 on cell proliferation, migration and multiple drug resistance 1 (MDR1) gene expression were determined in vitro. MG132 ameliorated intestinal inflammation of IL-10(-/-) mice by decreasing TNF-alpha mRNA expression in the colonic tissues, which was associated with suppression of NF-kappaB activation, and reduced significantly the number of Ki-67-positive intestinal epithelial cells. On the other hand, MG132 did not reduce intestinal inflammation in mice with DSS-induced colitis, and delayed significantly the recovery of body weight and epithelial regeneration. MG132 also suppressed significantly epithelial cell proliferation, cell migration and MDR1 gene expression in vitro. Proteasome inhibition reduces T cell-mediated intestinal inflammation, but may interrupt both epithelial regeneration and barrier function of colonic mucosa. Optimal use of proteasome inhibitor should be kept in mind when we consider its clinical application for patients with IBD.
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Affiliation(s)
- S Inoue
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Abstract
Altered transforming growth factor-beta (TGFbeta) expression may contribute to inflammatory bowel disease and modulate epithelial cell restitution. Interference with TGFbeta-mediated signaling inhibits excisional skin wound healing, but accelerates healing of incisional cutaneous wounds and wounds in some other tissues. Therefore, we sought to clarify the potential role of Smad3-dependent TGFbeta signaling in intestinal mucosal healing in Smad3 null mice. Jejunal serosal application of filter disks saturated with 75% acetic acid yielded a circumscribed reproducible ischemic mucosal ulcer 1 day later. We compared ulcer area at 3 and 5 days to day 1 in Smad3 knockout mice and syngeneic wild-type mice, and evaluated mucosal immunoreactivity at the ulcer edge for TGFbeta, phosphorylated (activated) focal adhesion kinase (pFAK), phosphorylated extracellular signal-related kinase (pERK), proliferating cell nuclear antigen and apoptosis by TUNEL. Ulcer healing in Smad3 null mice was 17% less at day 3 (n=14, P=0.022) and 15% less at day 5 (n=14, P=0.004) than in wild-type littermates. In wild-type mice, pFAK, pERK and TGFbeta immunoreactivity were elevated in epithelium immediately adjacent to the ulcer compared with more distant mucosa. However, this pattern of immunoreactivity for pFAK, pERK and TGFbeta was not observed in Smad3 null mice. Smad3 null mice exhibited increased epithelial proliferation and no differences in apoptotic cell death compared with wild types, suggesting that ulcer healing may reflect differences in restitutive cell migration. Thus, Smad3-dependent disruption of the TGFbeta signaling pathway impairs the healing of murine intestinal mucosal ulcers and alters patterns of activated FAK and ERK immunoreactivity important for cell migration at the ulcer edge. These studies suggest a significant role for Smad3-dependent TGFbeta signaling in intestinal mucosal healing.
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Bulut K, Felderbauer P, Deters S, Hoeck K, Schmidt-Choudhury A, Schmidt WE, Hoffmann P. Sensory neuropeptides and epithelial cell restitution: the relevance of SP- and CGRP-stimulated mast cells. Int J Colorectal Dis 2008; 23:535-41. [PMID: 18274763 DOI: 10.1007/s00384-008-0447-7] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 01/22/2008] [Indexed: 02/04/2023]
Abstract
BACKGROUND Calcitonin-gene-related peptide (CGRP) and substance P (SP) are neurotransmitters of extrinsic primary afferent neurons located within the dorsal root ganglia. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on intestinal mucosa was presumed. The mucosal protection partly depends on a CGRP-mediated modulation of mucosal blood flow. Limited data are available regarding CGRP- or SP-mediated effects on epithelial cell restitution. Having shown earlier that SP-stimulated fibroblasts but not CGRP-stimulated fibroblasts induce epithelial cell migration in vitro, the aim of this study was to explore whether mast cells mediate effects of SP and CGRP on epithelial cell restitution in vitro. METHODS Mast cells (C57) were exposed to SP [10(-12)-10(-6 M)] and CGRP [10(-12)-10(-7 M)]. After a 24-h incubation period, the cell supernatants (conditioned media, CDM) were taken from mast cell cultures and directly applied to rat intestinal epithelial cell lines-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h prior to the experiments. Epithelial cell migration was assessed by counting cells across the wound edge and epithelial cell proliferation was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide test. RESULTS CGRP significantly induced epithelial cell migration and proliferation via mast cells when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralizing anti-transforming growth factor-beta (TGF-beta) had been added to the cell cultures. SP had no effects on epithelial cells following stimulation of mast cells. CONCLUSION CGRP modulates epithelial cell restitution in vitro mediated by mast cells. The CGRP- and mast-cell-induced epithelial cell migration is TGF-beta dependent. This observation underlines an important role for extrinsic primary afferent neurons in mucosal defence and repair and in keeping the mucosal homeostasis. This knowledge leads to a better understanding of the interaction of the enteric nervous system and wound healing and may, in the future, lead to new therapeutic approaches to inflammatory diseases of the intestine.
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Affiliation(s)
- Kerem Bulut
- Department of Internal Medicine I, St. Josef Hospital, Ruhr-University of Bochum, Gudrun Str. 54, 44791, Bochum, Germany
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Bulut K, Pennartz C, Felderbauer P, Meier JJ, Banasch M, Bulut D, Schmitz F, Schmidt WE, Hoffmann P. Glucagon like peptide-2 induces intestinal restitution through VEGF release from subepithelial myofibroblasts. Eur J Pharmacol 2008; 578:279-85. [DOI: 10.1016/j.ejphar.2007.08.044] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2007] [Revised: 08/21/2007] [Accepted: 08/24/2007] [Indexed: 11/16/2022]
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Felderbauer P, Bulut K, Hoeck K, Deters S, Schmidt WE, Hoffmann P. Substance P induces intestinal wound healing via fibroblasts--evidence for a TGF-beta-dependent effect. Int J Colorectal Dis 2007; 22:1475-80. [PMID: 17520266 DOI: 10.1007/s00384-007-0321-z] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 04/20/2007] [Indexed: 02/04/2023]
Abstract
BACKGROUND Substance P (SP) and calcitonin gene-related peptide (CGRP) are neurotransmitters of the afferent sensory nervous system. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on the intestinal mucosa was presumed. In part, mucosal protection depends on a SP-mediated and CGRP-mediated modulation of mucosal blood flow after injury. We thought to explore whether there is a fibroblast-mediated effect of SP and CGRP on epithelial cell restitution in vitro. MATERIALS AND METHODS Rat kidney fibroblast (NRK-49F) cell lines were exposed to CGRP or SP in various concentrations. After incubation, the cell culture supernatants were taken from the fibroblast cultures and were directly applied to IEC-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h before the experiments. Epithelial cell migration was assessed by counting cells across the wound edge. Epithelial cell proliferation was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) test. RESULTS SP significantly induced epithelial cell migration and inhibited epithelial cell proliferation via stimulation of fibroblasts when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralising anti-transforming growth factor-beta (TGF-beta) was added to the cell cultures. CGRP had no effect on epithelial cells via stimulation of fibroblasts. Neither CGRP nor SP had any effect on epithelial cell migration or proliferation when directly applied to epithelial cells. CONCLUSION SP modulates epithelial cell restitution in vitro mediated by fibroblasts. The epithelial cell migration depends on a TGF-beta release from SP-stimulated fibroblasts. This observation underlines an important role for the sensory nervous system in mucosal defence and repair and in keeping mucosal homeostasis. Modulation of SP may be potentially useful for the treatment of various intestinal disorders characterised by injury and ineffective repair of the intestinal mucosa.
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Affiliation(s)
- Peter Felderbauer
- Department of Internal Medicine I, St. Josef-Hospital, Ruhr-University of Bochum, Gudrun Str. 54, 44791, Bochum, Germany.
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de Andrade Sá ER, Bitencourt B, Alvares EP, Gama P. In vivo effects of TGFbeta1 on the growth of gastric epithelium in suckling rats. ACTA ACUST UNITED AC 2007; 146:293-302. [PMID: 18068234 DOI: 10.1016/j.regpep.2007.11.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2007] [Revised: 10/19/2007] [Accepted: 11/01/2007] [Indexed: 10/22/2022]
Abstract
As the content of Transforming Growth Factor-beta (TGFbeta) wanes in the milk of lactating rat, an increase in TGFbeta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGFbeta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGFbeta1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGFbeta1 while TbetaRII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p<0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p<0.05). Importantly, TGFbeta1 inhibited cell proliferation (p<0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p<0.05). Also, TGFbeta1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p<0.001), and by morphological features at 12 h (p<0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGFbeta1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TGFbeta1 in gastric growth during postnatal development.
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Affiliation(s)
- Eunice Ribeiro de Andrade Sá
- Department of Cell and Developmental Biology, Institute of Biomedical Sciences, Av Prof Lineu Prestes 1524 ICB I, University of São Paulo, São Paulo, SP, 05508-900, Brazil
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Zhang H, Li Y, Wang J, Lu B, Wang B, Li Q, Li J. Sequential observations show upregulation of TGF-beta1 at the early phase of chronic small bowel rejection in rats. Dig Dis Sci 2007; 52:3224-30. [PMID: 17406824 DOI: 10.1007/s10620-007-9752-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2006] [Accepted: 01/01/2007] [Indexed: 12/09/2022]
Abstract
Although TGF-beta1 is related to chronic rejection in various solid organs, its role remains controversial in the setting of small bowel transplantation. The aim of this study is to investigate the distinct role of TGF-beta1 and its receptors in the setting of chronic small bowel rejection. Sequential protocol biopsies were performed every 2 weeks from week 2 till week 8 after transplantation and recipients were sacrificed at week 16 as the end point. Gene expression was evaluated by SYBR Green I real-time polymerase chain reaction and protein was determined by immunohistochemistry. The gene expressions of TGF-beta1 and Type I receptor were upregulated and attained statistical significance at 4, 6 and 8 weeks in allografts. Dynamic expressions of TGF-beta1 and Type I receptor mRNA shown by sequential protocol biopsies support the view that TGF-beta1 signaling is involved in the pathogenesis of chronic small bowel rejection, especially at the early phase.
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Affiliation(s)
- Haiyun Zhang
- Medical College, Nanjing University, Nanjing 210093, China
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Lin X, Zamora PO, Takahashi K, Lui Y. Alleviation of experimental ulcerative colitis with the synthetic peptide, F2A4-K-NS (Fibratide). Dig Dis Sci 2007; 52:2054-62. [PMID: 17404850 DOI: 10.1007/s10620-006-9641-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/12/2006] [Accepted: 10/09/2006] [Indexed: 12/09/2022]
Abstract
Recombinant fibroblast growth factors (FGFs) maintain the integrity of the gut epithelium and reduce mucosal injury in experimental inflammatory bowel disease (IBD). Chemically synthesized FGF mimetics could potentially extend the utility of FGFs by tailoring them for optimal bioactivity and oral administration, for example. Here, F2A4-K-NS (Fibratide), a synthetic FGF mimetic peptide, alleviated dextran sulfate sodium (DSS)-induced ulcerative colitis in mice when delivered systemically and, to a lesser extent, orally. Intraperitoneal injection of Fibratide (1 or 5 mg/kg/day) ameliorated DSS-induced ulcerative colitis, resulting in reduced weight loss, decreased colon wall thickening, and increased colon length. Fibratide also improved epithelial integrity by reducing histological-detectable crypt damage and inflammation. Orally administered Fibratide (1 mg/kg/day) was also effective in ameliorating symptoms with effects generally similar to those of intraperitoneal injection. In vitro studies were conducted to help clarify how Fibratide might act in vivo. Fibratide exhibited a modest enhancement of epithelial cell proliferation. On the other hand, Fibratide doubled the rate of epithelial cells migration and restitution in a cell culture model of wound repair. Collectively, the results indicate that Fibratide reduced the severity of experimental ulcerative colitis and may be potentially useful in the treatment of IBD.
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Affiliation(s)
- Xinhua Lin
- BioSurface Engineering Technologies, Inc., 9430 Key West Avenue, Suite 220, Rockville, Maryland 20850, USA.
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Karrasch T, Kim JS, Jang BI, Jobin C. The flavonoid luteolin worsens chemical-induced colitis in NF-kappaB(EGFP) transgenic mice through blockade of NF-kappaB-dependent protective molecules. PLoS One 2007; 2:e596. [PMID: 17611628 PMCID: PMC1895919 DOI: 10.1371/journal.pone.0000596] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2007] [Accepted: 06/11/2007] [Indexed: 12/21/2022] Open
Abstract
Background The flavonoid luteolin has anti-inflammatory properties both in vivo and in vitro. However, the impact of luteolin on experimental models of colitis is unknown. Methodology/Principal Findings To address the therapeutic impact of luteolin, NF-κBEGFP transgenic mice were fed a chow diet containing 2% luteolin- or isoflavone-free control chow (AIN-76), and acute colitis was induced using 3% dextran sodium sulfate (DSS). Additionally, development of spontaneous colitis was evaluated in IL-10−/−;NF-κBEGFP transgenic mice fed 2% luteolin chow diet or control chow diet. Interestingly, NF-κBEGFP transgenic mice exposed to luteolin showed worse DSS-induced colitis (weight loss, histological scores) compared to control-fed mice, whereas spontaneous colitis in IL-10−/−;NF-κBEGFP mice was significantly attenuated. Macroscopic imaging of live resected colon showed enhanced EGFP expression (NF-κB activity) in luteolin-fed mice as compared to control-fed animals after DSS exposure, while cecal EGFP expression was attenuated in luteolin-fed IL-10−/− mice. Interestingly, confocal microscopy showed that EGFP positive cells were mostly located in the lamina propria and not in the epithelium. Caspase 3 activation was significantly enhanced whereas COX-2 gene expression was reduced in luteolin-fed, DSS-exposed NF-κBEGFP transgenic mice as assessed by Western blot and immunohistochemical analysis. In vitro, luteolin sensitized colonic epithelial HT29 cells to TNFα-induced apoptosis, caspase 3 activation, DNA fragmentation and reduced TNFα-induced C-IAP1, C-IAP2 and COX-2 gene expression. Conclusions/Significance We conclude that while luteolin shows beneficial effects on spontaneous colitis, it aggravates DSS-induced experimental colitis by blocking NF-κB-dependent protective molecules in enterocytes.
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Affiliation(s)
- Thomas Karrasch
- Department of Medicine and Center for GI Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Joo-Sung Kim
- Department of Medicine and Center for GI Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Byung Ik Jang
- Department of Medicine and Center for GI Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Christian Jobin
- Department of Medicine and Center for GI Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * To whom correspondence should be addressed. E-mail:
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