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Geropoulos G, Psarras K, Koimtzis G, Fornasiero M, Anestiadou E, Geropoulos V, Michopoulou A, Papaioannou M, Kouzi-Koliakou K, Galanis I. Knockout Genes in Bowel Anastomoses: A Systematic Review of Literature Outcomes. J Pers Med 2024; 14:553. [PMID: 38929776 PMCID: PMC11205243 DOI: 10.3390/jpm14060553] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2024] [Revised: 04/12/2024] [Accepted: 04/16/2024] [Indexed: 06/28/2024] Open
Abstract
BACKGROUND The intestinal wound healing process is a complex event of three overlapping phases: exudative, proliferative, and remodeling. Although some mechanisms have been extensively described, the intestinal healing process is still not fully understood. There are some similarities but also some differences compared to other tissues. The aim of this systematic review was to summarize all studies with knockout (KO) experimental models in bowel anastomoses, underline any recent knowledge, and clarify further the cellular and molecular mechanisms of the intestinal healing process. A systematic review protocol was performed. MATERIALS AND METHODS Medline, EMBASE, and Scopus were comprehensively searched. RESULTS a total of eight studies were included. The silenced genes included interleukin-10, the four-and-one-half LIM domain-containing protein 2 (FHL2), cyclooxygenase-2 (COX-2), annexin A1 (ANXA-1), thrombin-activatable fibrinolysis inhibitor (TAFI), and heparin-binding epidermal growth factor (HB-EGF) gene. Surgically, an end-to-end bowel anastomosis was performed in the majority of the studies. Increased inflammatory cell infiltration in the anastomotic site was found in IL-10-, annexin-A1-, and TAFI-deficient mice compared to controls. COX-1 deficiency showed decreased angiogenesis at the anastomotic site. Administration of prostaglandin E2 in COX-2-deficient mice partially improved anastomotic leak rates, while treatment of ANXA1 KO mice with Ac2-26 nanoparticles reduced colitis activity and increased weight recovery following surgery. CONCLUSIONS our findings provide new insights into improving intestinal wound healing by amplifying the aforementioned genes using appropriate gene therapies. Further research is required to clarify further the cellular and micromolecular mechanisms of intestinal healing.
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Affiliation(s)
- Georgios Geropoulos
- 2nd Department of Propaedeutic Surgery, Hippokration Hospital, School of Medicine, Aristotle University of Thessaloniki, 54642 Thessaloniki, Greece; (G.G.); (G.K.); (V.G.); (A.M.); (I.G.)
| | - Kyriakos Psarras
- 2nd Department of Propaedeutic Surgery, Hippokration Hospital, School of Medicine, Aristotle University of Thessaloniki, 54642 Thessaloniki, Greece; (G.G.); (G.K.); (V.G.); (A.M.); (I.G.)
| | - Georgios Koimtzis
- 2nd Department of Propaedeutic Surgery, Hippokration Hospital, School of Medicine, Aristotle University of Thessaloniki, 54642 Thessaloniki, Greece; (G.G.); (G.K.); (V.G.); (A.M.); (I.G.)
| | | | - Elissavet Anestiadou
- Fourth Surgical Department, School of Medicine, Aristotle University of Thessaloniki, 57010 Thessaloniki, Greece;
| | - Vasileios Geropoulos
- 2nd Department of Propaedeutic Surgery, Hippokration Hospital, School of Medicine, Aristotle University of Thessaloniki, 54642 Thessaloniki, Greece; (G.G.); (G.K.); (V.G.); (A.M.); (I.G.)
| | - Anna Michopoulou
- 2nd Department of Propaedeutic Surgery, Hippokration Hospital, School of Medicine, Aristotle University of Thessaloniki, 54642 Thessaloniki, Greece; (G.G.); (G.K.); (V.G.); (A.M.); (I.G.)
- Laboratory of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Maria Papaioannou
- Laboratory of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Kokkona Kouzi-Koliakou
- Laboratory of Histology and Embryology, Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Ioannis Galanis
- 2nd Department of Propaedeutic Surgery, Hippokration Hospital, School of Medicine, Aristotle University of Thessaloniki, 54642 Thessaloniki, Greece; (G.G.); (G.K.); (V.G.); (A.M.); (I.G.)
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Zhou L, Zhou W, Joseph AM, Chu C, Putzel GG, Fang B, Teng F, Lyu M, Yano H, Andreasson KI, Mekada E, Eberl G, Sonnenberg GF. Group 3 innate lymphoid cells produce the growth factor HB-EGF to protect the intestine from TNF-mediated inflammation. Nat Immunol 2022; 23:251-261. [PMID: 35102343 PMCID: PMC8842850 DOI: 10.1038/s41590-021-01110-0] [Citation(s) in RCA: 43] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Accepted: 12/02/2021] [Indexed: 12/20/2022]
Abstract
Tumor necrosis factor (TNF) drives chronic inflammation and cell death in the intestine, and blocking TNF is a therapeutic approach in inflammatory bowel disease (IBD). Despite this knowledge, the pathways that protect the intestine from TNF are incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3s) protect the intestinal epithelium from TNF-induced cell death. This occurs independent of interleukin-22 (IL-22), and we identify that ILC3s are a dominant source of heparin-binding epidermal growth factor-like growth factor (HB-EGF). ILC3s produce HB-EGF in response to prostaglandin E2 (PGE2) and engagement of the EP2 receptor. Mice lacking ILC3-derived HB-EGF exhibit increased susceptibility to TNF-mediated epithelial cell death and experimental intestinal inflammation. Finally, human ILC3s produce HB-EGF and are reduced from the inflamed intestine. These results define an essential role for ILC3-derived HB-EGF in protecting the intestine from TNF and indicate that disruption of this pathway contributes to IBD.
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Chu Y, Teng J, Feng P, Liu H, Wang F, Wang H. Dexmedetomidine Attenuates Hypoxia/Reoxygenation Injury of H9C2 Myocardial Cells by Upregulating miR-146a Expression via the MAPK Signal Pathway. Pharmacology 2021; 107:14-27. [PMID: 34718238 DOI: 10.1159/000506814] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2019] [Accepted: 02/26/2020] [Indexed: 11/19/2022]
Abstract
INTRODUCTION AND OBJECTIVE Dexmedetomidine (Dex) and a number of miRNAs contribute to ischemia/reperfusion injury. We aimed to explore the role of Dex and miR-146a on myocardial cells injured by hypoxia/reoxygenation (H/R). METHOD H9C2 cells were injured by H/R. Cell viability was tested using the cell counting kit-8. Lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels were determined using commercial kits. Flow cytometry was performed to determine apoptosis rate and reactive oxygen species (ROS) level. Protein and mRNA levels were assessed using Western blot and qPCR. RESULTS miR-146a expression and cell viability of H9C2 cells were downregulated under the circumstance of H/R injury. The tendency could be reversed by Dex, which could also upregulate SOD activity and decrease apoptosis, LDH activity, MDA, 78-kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP) levels of H9C2 cells. GRP78, CHOP levels, and cell viability were negatively modulated by miR-146a. Dex elevated cell viability, catalase, MnSOD, and NAD(P)H dehydrogenase (NQO1) levels but suppressed apoptosis rate, GRP78, and CHOP levels by increasing miR-146a expression and downregulating ROS, phosphorylation of p38, and extracellular signal-regulated kinases 1/2 levels. By using SB203580 (SB), the p38 mitogen-activated protein kinase (MAPK) inhibitor, Dex or the inhibition of miR-146 upregulated cell viability but downregulated GRP78 and CHOP levels. CONCLUSION Dex might regulate miR-146a expression, which could further modulate the endoplasmic reticulum stress and oxidative stress and eventually affect the cell viability and apoptosis of myocardial cells injured by H/R via the MAPK signal pathway.
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Affiliation(s)
- Yi Chu
- Department of Cardiology, The Second Affiliated Hospital of Air Force Military Medical University, Xi'an, China
| | - Jiwei Teng
- Department of Cardiology, The Second Affiliated Hospital of Air Force Military Medical University, Xi'an, China
| | - Pin Feng
- Department of Cardiology, The Second Affiliated Hospital of Air Force Military Medical University, Xi'an, China
| | - Hui Liu
- Department of Cardiology, The Second Affiliated Hospital of Air Force Military Medical University, Xi'an, China
| | - Fangfang Wang
- Department of Cardiology, The Second Affiliated Hospital of Air Force Military Medical University, Xi'an, China
| | - Haiyan Wang
- Department of Cardiology, The Second Affiliated Hospital of Air Force Military Medical University, Xi'an, China
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Human Milk Growth Factors and Their Role in NEC Prevention: A Narrative Review. Nutrients 2021; 13:nu13113751. [PMID: 34836007 PMCID: PMC8620589 DOI: 10.3390/nu13113751] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 10/05/2021] [Accepted: 10/09/2021] [Indexed: 12/25/2022] Open
Abstract
Growing evidence demonstrates human milk's protective effect against necrotizing enterocolitis (NEC). Human milk derives these properties from biologically active compounds that influence intestinal growth, barrier function, microvascular development, and immunological maturation. Among these protective compounds are growth factors that are secreted into milk with relatively high concentrations during the early postnatal period, when newborns are most susceptible to NEC. This paper reviews the current knowledge on human milk growth factors and their mechanisms of action relevant to NEC prevention. It will also discuss the stability of these growth factors with human milk pasteurization and their potential for use as supplements to infant formulas with the goal of preventing NEC.
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Wang ZY, Lin JY, Feng YR, Liu DS, Zhao XZ, Li T, Li SY, Sun JC, Li SF, Jia WY, Jing HR. Recombinant angiopoietin-like protein 4 attenuates intestinal barrier structure and function injury after ischemia/reperfusion. World J Gastroenterol 2021; 27:5404-5423. [PMID: 34539141 PMCID: PMC8409166 DOI: 10.3748/wjg.v27.i32.5404] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/24/2021] [Revised: 06/17/2021] [Accepted: 07/30/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Intestinal barrier breakdown, a frequent complication of intestinal ischemia-reperfusion (I/R) including dysfunction and the structure changes of the intestine, is characterized by a loss of tight junction and enhanced permeability of the intestinal barrier and increased mortality. To develop effective and novel therapeutics is important for the improvement of outcome of patients with intestinal barrier deterioration. Recombinant human angiopoietin-like protein 4 (rhANGPTL4) is reported to protect the blood-brain barrier when administered exogenously, and endogenous ANGPTL4 deficiency deteriorates radiation-induced intestinal injury.
AIM To identify whether rhANGPTL4 may protect intestinal barrier breakdown induced by I/R.
METHODS Intestinal I/R injury was elicited through clamping the superior mesenteric artery for 60 min followed by 240 min reperfusion. Intestinal epithelial (Caco-2) cells and human umbilical vein endothelial cells were challenged by hypoxia/ reoxygenation to mimic I/R in vitro.
RESULTS Indicators including fluorescein isothiocyanate-conjugated dextran (4 kilodaltons; FD-4) clearance, ratio of phosphorylated myosin light chain/total myosin light chain, myosin light chain kinase and loss of zonula occludens-1, claudin-2 and VE-cadherin were significantly increased after intestinal I/R or cell hypoxia/reoxygenation. rhANGPTL4 treatment significantly reversed these indicators, which were associated with inhibiting the inflammatory and oxidative cascade, excessive activation of cellular autophagy and apoptosis and improvement of survival rate. Similar results were observed in vitro when cells were challenged by hypoxia/reoxygenation, whereas rhANGPTL4 reversed the indicators close to normal level in Caco-2 cells and human umbilical vein endothelial cells significantly.
CONCLUSION rhANGPTL4 can function as a protective agent against intestinal injury induced by intestinal I/R and improve survival via maintenance of intestinal barrier structure and functions.
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Affiliation(s)
- Zi-Yi Wang
- Emergent Intensive Care Unit, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
| | - Jian-Yu Lin
- Department of Gastrointestinal Surgery, Weihai Central Hospital, Weihai 264200, Shandong Province, China
| | - Yang-Rong Feng
- Graduate College, Shandong First Medical University, Jinan 271000, Shandong Province, China
| | - De-Shun Liu
- Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, Liaoning Province, China
| | - Xu-Zi Zhao
- Department of Pharmacology, Dalian Medical University, Dalian 116044, Liaoning Province, China
| | - Tong Li
- Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100000, China
| | - Si-Yuan Li
- Department of General Surgery, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, Shandong Province, China
| | - Jing-Chao Sun
- Department of General Surgery, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, Shandong Province, China
| | - Shu-Feng Li
- Department of General Surgery, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, Shandong Province, China
| | - Wen-Yan Jia
- Physiological Examination Center, The Affiliated Hospital of Qingdao University, Qingdao 266000, Shandong Province, China
| | - Hui-Rong Jing
- Department of General Surgery, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, Shandong Province, China
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Wang M, Zhang J, Zhang J, Sun K, Li Q, Kuang B, Wang MMZ, Hou S, Gong N. Methyl eugenol attenuates liver ischemia reperfusion injury via activating PI3K/Akt signaling. Int Immunopharmacol 2021; 99:108023. [PMID: 34358859 DOI: 10.1016/j.intimp.2021.108023] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Revised: 07/20/2021] [Accepted: 07/23/2021] [Indexed: 12/11/2022]
Abstract
BACKGROUND Liver ischemia reperfusion injury (LIRI) often occurs during liver transplantation, resection, and various circulatory shock procedures, leading to severe metabolic disorders, inflammatory immune responses, oxidative stress injury, and cell apoptosis. Methyl eugenol (ME) is structurally similar to eugenol and has anti-inflammatory and apoptotic pharmacological effects. However, whether ME protects the liver from LIRI damage requires further investigation. METHODS We established a partially warm LIRI model by subjecting C57BL/6J mice to 60 min of ischemia, followed by reperfusion for 6 h. We also established a hypoxia-reoxygenation injury (H/R) cell model by subjecting AML12 (a mouse liver cell line) cells to 24 h hypoxia, followed by 18 h normoxia. The extent of liver injury was assessed by serum transaminase concentrations, hematoxylin and eosin staining, quantitative real-time PCR, myeloperoxidase activity, and TUNEL analysis. Apoptosis was detected using flow cytometry. The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-Bad, Bad, Bcl-2, Bax, and cleaved caspase-3 were detected by western blotting. LY294002, an inhibitor of PI3K/Akt signaling, was used to elucidate the relationship between ME and PI3K/Akt signaling. RESULTS ME successfully alleviated LIRI-induced liver injury, inflammatory response, and apoptosis induced, as well as liver cell injury induced by hypoxia reoxygenation. ME is known to activate the PI3K/Akt signaling pathway in hepatocyte injury in vivo and in vitro, and when this signaling pathway is inhibited, the protective effect of ME is abrogated. CONCLUSIONS The use of ME is a potential therapeutic approach for regulating LIRI by activating PI3K/Akt signaling.
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Affiliation(s)
- Mengqin Wang
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Ji Zhang
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Jiasi Zhang
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Kailun Sun
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Qingwen Li
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Baicheng Kuang
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - M M Zhiheng Wang
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Shuaiheng Hou
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China
| | - Nianqiao Gong
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, Wuhan, Hubei 430030, China.
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Chen Y, Song F, Tu M, Wu S, He X, Liu H, Xu C, Zhang K, Zhu Y, Zhou R, Jin C, Wang P, Zhang H, Tian M. Quantitative proteomics revealed extensive microenvironmental changes after stem cell transplantation in ischemic stroke. Front Med 2021; 16:429-441. [PMID: 34241786 DOI: 10.1007/s11684-021-0842-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Accepted: 11/24/2020] [Indexed: 12/28/2022]
Abstract
The local microenvironment is essential to stem cell-based therapy for ischemic stroke, and spatiotemporal changes of the microenvironment in the pathological process provide vital clues for understanding the therapeutic mechanisms. However, relevant studies on microenvironmental changes were mainly confined in the acute phase of stroke, and long-term changes remain unclear. This study aimed to investigate the microenvironmental changes in the subacute and chronic phases of ischemic stroke after stem cell transplantation. Herein, induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) were transplanted into the ischemic brain established by middle cerebral artery occlusion surgery. Positron emission tomography imaging and neurological tests were applied to evaluate the metabolic and neurofunctional alterations of rats transplanted with stem cells. Quantitative proteomics was employed to investigate the protein expression profiles in iPSCs-transplanted brain in the subacute and chronic phases of stroke. Compared with NSCs-transplanted rats, significantly increased glucose metabolism and neurofunctional scores were observed in iPSCs-transplanted rats. Subsequent proteomic data of iPSCs-transplanted rats identified a total of 39 differentially expressed proteins in the subacute and chronic phases, which are involved in various ischemic stroke-related biological processes, including neuronal survival, axonal remodeling, antioxidative stress, and mitochondrial function restoration. Taken together, our study indicated that iPSCs have a positive therapeutic effect in ischemic stroke and emphasized the wide-ranging microenvironmental changes in the subacute and chronic phases.
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Affiliation(s)
- Yao Chen
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China.,Department of Radiology, Zhejiang Hospital, Hangzhou, 310030, China
| | - Fahuan Song
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Mengjiao Tu
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China.,Department of PET Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China
| | - Shuang Wu
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Xiao He
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Hao Liu
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Caiyun Xu
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Kai Zhang
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Yuankai Zhu
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Rui Zhou
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Chentao Jin
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China.,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China.,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China
| | - Ping Wang
- Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, 310027, China.,College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, 310027, China
| | - Hong Zhang
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China. .,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China. .,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China. .,Shanxi Medical University, Taiyuan, 030001, China. .,Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, 310027, China. .,College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, 310027, China.
| | - Mei Tian
- Department of Nuclear Medicine and Medical PET Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China. .,Institute of Nuclear Medicine and Molecular Imaging, Zhejiang University, Hangzhou, 310009, China. .,Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou, 310009, China.
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Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF. Int J Mol Sci 2020; 21:ijms21134755. [PMID: 32635412 PMCID: PMC7370175 DOI: 10.3390/ijms21134755] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 06/30/2020] [Accepted: 07/02/2020] [Indexed: 12/14/2022] Open
Abstract
Maintaining intestinal health in livestock is critical during the weaning period. The precise mechanisms of intestinal dysfunction during this period are not fully understood, although these can be alleviated by phlorotannins, including eckol. This question was addressed by evaluating the changes in gene expression and intestinal function after eckol treatment during suckling-to-weaning transition. The biological roles of differentially expressed genes (DEGs) in intestinal development were investigated by assessing intestinal wound healing and barrier functions, as well as the associated signaling pathways and oxidative stress levels. We identified 890 DEGs in the intestine, whose expression was altered by eckol treatment, including pancreatic and duodenal homeobox (PDX)1, which directly regulate heparin-binding epidermal growth factor-like growth factor (HBEGF) expression in order to preserve intestinal barrier functions and promote wound healing through phosphoinositide 3-kinase (PI3K)/AKT and P38 signaling. Additionally, eckol alleviated H2O2-induced oxidative stress through PI3K/AKT, P38, and 5’-AMP-activated protein kinase (AMPK) signaling, improved growth, and reduced oxidative stress and intestinal permeability in pigs during the weaning period. Eckol modulates intestinal barrier functions, wound healing, and oxidative stress through PDX/HBEGF, and improves growth during the suckling-to-weaning transition. These findings suggest that eckol can be used as a feed supplement in order to preserve the intestinal functions in pigs and other livestock during this process.
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Zhang HY, Wang F, Chen X, Meng X, Feng C, Feng JX. Dual roles of commensal bacteria after intestinal ischemia and reperfusion. Pediatr Surg Int 2020; 36:81-91. [PMID: 31541279 DOI: 10.1007/s00383-019-04555-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 09/13/2019] [Indexed: 11/30/2022]
Abstract
PURPOSE The roles of commensal bacteria after intestinal ischemia and reperfusion (IIR) are unclear. In current study, we aim to investigate the effects and underlying mechanisms of commensal bacteria in injury and epithelial restitution after IIR. METHODS Commensal gut bacteria were deleted by broad-spectrum antibiotics in mice. IIR was induced by clamping superior mesenteric artery. Intestinal injury, permeability, epithelial proliferation, and proinflammatory activity of mesenteric lymph were investigated. RESULTS Commensals deletion improved mice survival in the early phase, but failed to improve the overall survival at 96 h after IIR. Commensals deletion reduced proliferation of intestinal epithelial cells (IEC) and augmented proinflammatory activity of mesenteric lymph after IIR. Lipopolysaccharides (LPS) supplement promoted IEC proliferation and improved survival in mice with commensals deletion after IIR. LPS induced production of prostaglandin E2 (PGE2) in mucosa via toll-like receptor 4-NFκB-cyclooxygenase 2 pathway. PGE2 enhanced IEC proliferation in vivo, which was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2. Blocking of EGFR, PI3K/Akt activity abolished LPS-induced IEC proliferation. CONCLUSIONS Commensal bacteria are essential for epithelial restitution after IIR, which enhance IEC proliferation via induction of PGE2.
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Affiliation(s)
- Hong-Yi Zhang
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave, Wuhan, 430030, China
| | - Fang Wang
- Department of Neurology, The Central Hospital of Wuhan, Huazhong University of Science and Technology, Wuhan, 430014, China
| | - Xuyong Chen
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave, Wuhan, 430030, China
| | - Xinrao Meng
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave, Wuhan, 430030, China
| | - Chenzhao Feng
- Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Jie-Xiong Feng
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave, Wuhan, 430030, China.
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10
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Zu G, Guo J, Zhou T, Che N, Liu B, Wang D, Zhang X. The transcription factor FoxM1 activates Nurr1 to promote intestinal regeneration after ischemia/reperfusion injury. Exp Mol Med 2019; 51:1-12. [PMID: 31704909 PMCID: PMC6841953 DOI: 10.1038/s12276-019-0343-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2019] [Revised: 07/12/2019] [Accepted: 08/07/2019] [Indexed: 12/22/2022] Open
Abstract
FoxM1 is involved in the regeneration of several organs after injury and expressed in the intestinal mucosa. The intrinsic mechanism of FoxM1 activity in the mucosa after intestinal ischemia/reperfusion (I/R) injury has not been reported. Therefore, we investigated the role of FoxM1 in mediating intestinal mucosa regeneration after I/R injury. Expression of FoxM1 and the proliferation of intestinal mucosa epithelial cells were examined in rats with intestinal I/R injury and an IEC-6 cell hypoxia/reperfusion (H/R) model. The effects of FoxM1 inhibition or activation on intestinal epithelial cell proliferation were measured. FoxM1 expression was consistent with the proliferation of intestinal epithelial cells in the intestinal mucosa after I/R injury. Inhibition of FoxM1 expression led to the downregulation of Ki-67 expression mediated by the inhibited expression of Nurr1, and FoxM1 overexpression promoted IEC-6 cell proliferation after H/R injury through activating Nurr1 expression. Furthermore, FoxM1 directly promoted the transcription of Nurr1 by directly binding the promoter of Nurr1. Further investigation showed low expression levels of FoxM1, Nurr1, and Ki-67 in the intestinal epithelium of patients with intestinal ischemic injury. FoxM1 acts as a critical regulator of intestinal regeneration after I/R injury by directly promoting the transcription of Nurr1. The FoxM1/Nurr1 signaling pathway represents a promising therapeutic target for intestinal I/R injury and related clinical diseases. A signaling pathway that promotes the regeneration of intestinal cells in rats represents a promising therapeutic target for treating intestinal injury in humans. A team led by Guo Zu and Jing Guo from Dalian Medical University in China investigated the role of a regulatory protein called FoxM1 in repairing intestinal damage after a period of inadequate blood flow to the tissues of the gastrointestinal tract. They showed in rat models that FoxM1 promoted the proliferation of intestinal cells after injury by activating other proteins in a particular signaling pathway. Looking at tissue samples from five people who experienced intestinal injury as a result of restricted blood flow, the researchers detected low expression levels of FoxM1 and its downstream signaling intermediaries. Boosting the activity of those proteins could help promote healing and regeneration.
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Affiliation(s)
- Guo Zu
- Department of Gastroenterology Surgery, The Dalian Municipal Central Hospital Affiliated of Dalian Medical University, 116033, Dalian, China.
| | - Jing Guo
- Institute of Integrative Medicine, Dalian Medical University, 116044, Dalian, China
| | - Tingting Zhou
- Department of Neurology, The First Affiliated Hospital of Dalian Medical University, 116011, Dalian, China
| | - Ningwei Che
- Department of Neurosurgery, The Second Affiliated Hospital of Dalian Medical University, 116027, Dalian, China
| | - Baiying Liu
- Department of Gastroenterology Surgery, The Dalian Municipal Central Hospital Affiliated of Dalian Medical University, 116033, Dalian, China
| | - Dong Wang
- Department of Gastroenterology Surgery, The Dalian Municipal Central Hospital Affiliated of Dalian Medical University, 116033, Dalian, China
| | - Xiangwen Zhang
- Department of Gastroenterology Surgery, The Dalian Municipal Central Hospital Affiliated of Dalian Medical University, 116033, Dalian, China
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11
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Kong T, Liu M, Ji B, Bai B, Cheng B, Wang C. Role of the Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway in Ischemia-Reperfusion Injury. Front Physiol 2019; 10:1038. [PMID: 31474876 PMCID: PMC6702336 DOI: 10.3389/fphys.2019.01038] [Citation(s) in RCA: 56] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2019] [Accepted: 07/29/2019] [Indexed: 12/11/2022] Open
Abstract
Extracellular signal-regulated kinase 1/2 (ERK1/2), an important member of the mitogen-activated protein kinase family, is found in many organisms, and it participates in intracellular signal transduction. Various stimuli induce phosphorylation of ERK1/2 in vivo and in vitro. Phosphorylated ERK1/2 moves to the nucleus, activates many transcription factors, regulates gene expression, and controls various physiological processes, finally inducing repair processes or cell death. With the aging of the population around the world, the occurrence of ischemia-reperfusion injury (IRI), especially in the brain, heart, kidney, and other important organs, is becoming increasingly serious. Abnormal activation of the ERK1/2 signaling pathway is closely related to the development and the metabolic mechanisms of IRI. However, the effects of this signaling pathway and the underlying mechanism differ between various models of IRI. This review summarizes the ERK1/2 signaling pathway and the molecular mechanism underlying its role in models of IRI in the brain, heart, liver, kidneys, and other organs. This information will help to deepen the understanding of ERK1/2 signals and deepen the exploration of IRI treatment based on the ERK1/2 study.
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Affiliation(s)
- Tingting Kong
- Cheeloo College of Medicine, Shandong University, Jinan, China.,School of Mental Health, Neurobiology Key Laboratory of Jining Medical University in Colleges of Shandong, Jining Medical University, Jining, China
| | - Minghui Liu
- School of Mental Health, Neurobiology Key Laboratory of Jining Medical University in Colleges of Shandong, Jining Medical University, Jining, China
| | - Bingyuan Ji
- School of Mental Health, Neurobiology Key Laboratory of Jining Medical University in Colleges of Shandong, Jining Medical University, Jining, China
| | - Bo Bai
- School of Mental Health, Neurobiology Key Laboratory of Jining Medical University in Colleges of Shandong, Jining Medical University, Jining, China
| | - Baohua Cheng
- School of Mental Health, Neurobiology Key Laboratory of Jining Medical University in Colleges of Shandong, Jining Medical University, Jining, China
| | - Chunmei Wang
- School of Mental Health, Neurobiology Key Laboratory of Jining Medical University in Colleges of Shandong, Jining Medical University, Jining, China
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12
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Shelby RD, Cromeens B, Rager TM, Besner GE. Influence of Growth Factors on the Development of Necrotizing Enterocolitis. Clin Perinatol 2019; 46:51-64. [PMID: 30771819 PMCID: PMC6380490 DOI: 10.1016/j.clp.2018.10.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Growth factors have important roles in gastrointestinal tract development, maintenance, and response to injury. Various experiments have been used to demonstrate growth factor influence in multiple disease processes. These studies demonstrated enhancement of mucosal proliferation, intestinal motility, immune modulation, and many other beneficial effects. Select growth factors, including epidermal growth factor and heparin-binding epidermal growth factor like growth factor, demonstrate some beneficial effects in experimental and clinical intestinal injury demonstrated in necrotizing enterocolitis. The roles of glucagon-like peptide 2, insulin-like growth factor 1, erythropoietin, growth hormone, and hepatocyte growth factor in necrotizing enterocolitis are summarized in this article.
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Affiliation(s)
- Rita D. Shelby
- Surgical Research Fellow, Department of Pediatric Surgery, Nationwide Children’s Hospital, Center for Perinatal Research, the Research Institute at Nationwide Children’s Hospital, The Ohio State University College of Medicine, Columbus, OH
| | - Barrett Cromeens
- Surgical Research Fellow, Department of Pediatric Surgery, Nationwide Children’s Hospital, Center for Perinatal Research, the Research Institute at Nationwide Children’s Hospital, The Ohio State University College of Medicine, Columbus, OH
| | - Terrance M Rager
- Surgical Research Fellow, Department of Pediatric Surgery, Nationwide Children’s Hospital, Center for Perinatal Research, the Research Institute at Nationwide Children’s Hospital, The Ohio State University College of Medicine, Columbus, OH
| | - Gail E. Besner
- Chief, Department of Pediatric Surgery, H. William Clatworthy, Jr. Professor of Surgery, Department of Pediatric Surgery, Nationwide Children’s Hospital, Center for Perinatal Research, the Research Institute at Nationwide Children’s Hospital, The Ohio State University College of Medicine, Columbus, OH
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13
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Li Q, Cui S, Jing G, Ding H, Xia Z, He X. The role of PI3K/Akt signal pathway in the protective effects of propofol on intestinal and lung injury induced by intestinal ischemia/reperfusion1. Acta Cir Bras 2019; 34:e20190010000005. [PMID: 30785506 PMCID: PMC6585923 DOI: 10.1590/s0102-865020190010000005] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Accepted: 12/15/2019] [Indexed: 12/15/2022] Open
Abstract
Purpose To investigate the role of PI3k/Akt signal pathway in the protective effects
of propofol on intestinal and lung injury induced by intestinal
ischemia/reperfusion(I/R). Methods Male Sprague-Dawley rats were subjected to 45 min of ischemia by occluding
the superior mesenteric artery and to 2h of reperfusion to establish the
model of I/R. Twenty four rats were randomly divided into four groups: Sham,
intestinal I/R (II/R), propofol (P), wortmannin (W). In groups P, W,
propofol was injected intravenously and continuously at the onset of
reperfusion via infusion pump. PI3K inhibitor (wortmannin) was administered
intravenously in group W 25 min before ischemia. Intestinal tissues and lung
tissues were obtained for determination of histologic injury, wet/dry weight
ratio, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and
myeloperoxidase (MPO) activities. Meanwhile, the expressions of caspase-3
and phosphorylated Akt (p-Akt) in intestines and lungs were detected by
western blot. Results Propofol treatment alleviated intestinal and lung morphological changes which
were observed in II/R group , Moreover, wet/dry weight ratio, the MDA level,
MPO activity and expression of caspase-3 were significantly decreased
whereas the SOD activity and p-Akt expression were significantly increased.
Notably, the protections were significantly reversed by pretreatment of
wortmannin. Conclusion: PI3K/Akt pathway activation play a critical role in the protective effects of
propofol on intestinal and lung injury induced by ischemia/reperfusion.
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Affiliation(s)
- Qingwen Li
- MD, Department of Anesthesiology, Renmin Hospital of Wuhan University, Hubei, China. Manuscript preparation and writng
| | - Shanshan Cui
- MD, Department of Anesthesiology, Renmin Hospital of Wuhan University, Hubei, China. Conception and design of the study, acquisiton of data
| | - Guoqing Jing
- MD, Department of Anesthesiology, Renmin Hospital of Wuhan University, Hubei, China. Analysis and interpretation of data, technical procedures
| | - Huang Ding
- MD, Department of Anesthesiology, Renmin Hospital of Wuhan University, Hubei, China. Histopathological examinations, statistics analysis
| | - Zhongyuan Xia
- MD, Department of Anesthesiology, Renmin Hospital of Wuhan University, Hubei, China. Final approval
| | - Xianghu He
- MD, Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Hubei, China. Critical revision
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14
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Li P, Deng Q, Liu J, Yan J, Wei Z, Zhang Z, Liu H, Li B. Roles for HB-EGF in Mesenchymal Stromal Cell Proliferation and Differentiation During Skeletal Growth. J Bone Miner Res 2019; 34:295-309. [PMID: 30550637 PMCID: PMC7816091 DOI: 10.1002/jbmr.3596] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2018] [Revised: 09/04/2018] [Accepted: 09/22/2018] [Indexed: 12/22/2022]
Abstract
HB-EGF, a member of the EGF superfamily, plays important roles in development and tissue regeneration. However, its functions in skeletal stem cells and skeleton development and growth remain poorly understood. Here, we used the Cre/LoxP system to ablate or express HB-EGF in Dermo1+ mesenchymal stromal cells and their progenies, including chondrocytes and osteoblast lineage cells, and bone marrow stromal cells (BMSCs). Dermo1-Cre; HB-EGFf/f mice only showed a modest increase in bone mass, whereas Dermo1-HB-EGF mice developed progressive chondrodysplasia, chondroma, osteoarthritis-like joint defects, and loss of bone mass and density, which were alleviated by treatment with EGFR inhibitor AG1478. The cartilage defects were recapitulated in chondrocyte-specific HB-EGF overexpression (Col2-HB-EGF) mice with a lesser severity. Dermo1-HB-EGF mice showed an increase in proliferation but defects in differentiation of chondrocytes and osteoblasts. HB-EGF promoted BMSC proliferation via the Akt1 and Erk pathways but inhibited BMSC differentiation via restraining Smad1/5/8 activation. However, Dermo1-HB-EGF mice showed normal osteoclastogenesis and bone resorption. These results reveal an important function of autocrine or paracrine HB-EGF in mesenchymal stromal cell proliferation and differentiation and suggest that EGF signaling needs to be tightly controlled to maintain bone and articular cartilage integrity. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
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Affiliation(s)
- Ping Li
- Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Qi Deng
- Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Jiajia Liu
- Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Jianshe Yan
- School of Life Sciences, Shanghai University, Shanghai, China.,Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Zhanying Wei
- Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Key Clinical Center for Metabolic Disease, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
| | - Zhenlin Zhang
- Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Key Clinical Center for Metabolic Disease, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
| | - Huijuan Liu
- Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Baojie Li
- Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
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15
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N-acetylcysteine modulates lipopolysaccharide-induced intestinal dysfunction. Sci Rep 2019; 9:1004. [PMID: 30700808 PMCID: PMC6353963 DOI: 10.1038/s41598-018-37296-x] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2018] [Accepted: 11/24/2018] [Indexed: 12/21/2022] Open
Abstract
The gastrointestinal epithelium functions in nutrient absorption and pathogens barrier and its dysfunction directly affects livestock performance. N-Acetylcysteine (NAC) improves mucosal function, but its effects on intestinal functions at the molecular level remain unclear. Here, we performed gene expression profiling of the pig small intestine after dietary NAC treatment under LPS challenge and investigated the effects of NAC on intestinal epithelial cells in vitro. Dietary NAC supplementation under LPS challenge altered the small intestine expression of 959 genes related to immune response, inflammatory response, oxidation-reduction process, cytokine-cytokine receptor interaction, and the cytokine-mediated signalling, Toll-like receptor signalling pathway, Jak-STAT signalling pathway, and TNF signalling pathway. We then analysed the expression patterns of the top 10 altered genes, and found that NAC markedly stimulated HMGCS3 and LDHC expression in IPEC-J2 cells. In vitro, NAC pre-treatment significantly reduced TNF-α and NF-κB, TNF-α, IFN-γ, and IL-6 expression in LPS-induced IPEC-J2 cells. NAC treatment also significantly reduced oxidative stress in LPS-induced IPEC-J2 cells and alleviated intestinal barrier function and wound healing. Thus, NAC as a feed additive can enhance livestock intestinal health by modulating intestinal inflammation, permeability, and wound healing under LPS-induced dysfunction, improving our molecular understanding of the effects of NAC on the intestine.
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16
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Dao DT, Anez-Bustillos L, Adam RM, Puder M, Bielenberg DR. Heparin-Binding Epidermal Growth Factor-Like Growth Factor as a Critical Mediator of Tissue Repair and Regeneration. THE AMERICAN JOURNAL OF PATHOLOGY 2018; 188:2446-2456. [PMID: 30142332 PMCID: PMC6207098 DOI: 10.1016/j.ajpath.2018.07.016] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/02/2018] [Revised: 06/21/2018] [Accepted: 07/13/2018] [Indexed: 11/20/2022]
Abstract
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family. It contains an EGF-like domain as well as a heparin-binding domain that allows for interactions with heparin and cell-surface heparan sulfate. Soluble mature HB-EGF, a ligand of human epidermal growth factor receptors 1 and 4, is cleaved from the membrane-associated pro-HB-EGF by matrix metalloproteinase or a disintegrin and metalloproteinase in a process called ectodomain shedding. Signaling through human epidermal growth factor receptors 1 and 4 results in a variety of effects, including cellular proliferation, migration, adhesion, and differentiation. HB-EGF levels increase in response to different forms of injuries as well as stimuli, such as lysophosphatidic acid, retinoic acid, and 17β-estradiol. Because it is widely expressed in many organs, HB-EGF plays a critical role in tissue repair and regeneration throughout the body. It promotes cutaneous wound healing, hepatocyte proliferation after partial hepatectomy, intestinal anastomosis strength, alveolar regeneration after pneumonectomy, neurogenesis after ischemic injury, bladder wall thickening in response to urinary tract obstruction, and protection against ischemia/reperfusion injury to many cell types. Additionally, innovative strategies to deliver HB-EGF to sites of organ injury or to increase the endogenous levels of shed HB-EGF have been attempted with promising results. Harnessing the reparatory properties of HB-EGF in the clinical setting, therefore, may produce therapies that augment the treatment of various organ injuries.
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Affiliation(s)
- Duy T Dao
- Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Lorenzo Anez-Bustillos
- Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Rosalyn M Adam
- Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Urological Diseases Research Center, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Mark Puder
- Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Diane R Bielenberg
- Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.
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17
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Paveljšek D, Juvan P, Košir R, Rozman D, Hacin B, Ivičak-Kocjan K, Rogelj I. Lactobacillus fermentum L930BB and Bifidobacterium animalis subsp. animalis IM386 initiate signalling pathways involved in intestinal epithelial barrier protection. Benef Microbes 2018; 9:515-525. [PMID: 29633647 DOI: 10.3920/bm2017.0107] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The manipulation of intestinal microbiota with beneficial microbes represents a promising alternative or adjunct therapy in gastrointestinal disorders and inflammation. The current study aims to clarify the signalling pathways and evaluate the possible beneficial effects of the combination of two strains. We used a dextran sulphate sodium (DSS)-induced mouse model of colitis. RNA extracted from the middle part of the colon tissue was used for examination of the global gene expression with Affymetrix microarrays. An enrichment analysis of the KEGG pathways was performed, and a subset of genes associated with intestinal epithelial barrier function was verified with qPCR. A clinical condition assessment of the differently treated mice revealed that the combination of these two bacterial strains was safe for use as a dietary supplement. All animals treated with DSS had affected colons and suffered weight loss. There were very small differences between the diseased groups, although the depth of inflammation was lower when cyclosporine A or the strain mixture was used. We discovered that the prophylactic administration of the Lactobacillus fermentum L930BB (L930BB) and Bifidobacterium animalis subsp. animalis IM386 (IM386) strains led to an anti-apoptotic pathway through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and to the activation of pathways involved in the regulation of actin cytoskeleton via protein kinase C and GTPases. Reorganisation of actin cytoskeleton and decreased apoptosis are both helpful in intestinal epithelial cell reconstitution. We confirm important previous observations, showing that these pathways are downstream targets of Toll-like receptor 2 and fibroblast growth factor initiated signalling. Taken together, these results suggest that the combination of L930BB and IM386 could aid in the regeneration of the intestinal epithelium during pathogenesis via pattern recognition receptors and the stimulation of growth factor synthesis.
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Affiliation(s)
- D Paveljšek
- 1 Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia
| | - P Juvan
- 2 Centre for Functional Genomics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia
| | - R Košir
- 2 Centre for Functional Genomics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia.,3 BIA Separations CRO, Labena d.o.o., Verovškova 64, 1000 Ljubljana, Slovenia
| | - D Rozman
- 2 Centre for Functional Genomics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia
| | - B Hacin
- 4 National Veterinary Institute, Veterinary Faculty, University of Ljubljana, Pri Hrastu 18, 5000 Nova Gorica, Slovenia
| | - K Ivičak-Kocjan
- 5 Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova ulica 19, 1000 Ljubljana, Slovenia
| | - I Rogelj
- 1 Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia
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18
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A Novel Role of Spred2 in the Colonic Epithelial Cell Homeostasis and Inflammation. Sci Rep 2016; 6:37531. [PMID: 27869219 PMCID: PMC5116627 DOI: 10.1038/srep37531] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Accepted: 10/27/2016] [Indexed: 12/13/2022] Open
Abstract
Rapid and adequate mucosal healing is important for a remission of ulcerative colitis (UC) patients. Here, we examined whether Spred2, a member of the Sprouty-related EVH1-domain-containing proteins that inhibit the Ras/Raf/ERK pathway, plays a role in colonic mucosal homeostasis and inflammation by using Spred2 knockout (KO) mice. We first detected increased epithelial cell proliferation and cadherin 1 expression in the colon of naïve Spred2 KO mice compared to wild-type mice. Interestingly, Spred2 KO mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis as indicated by lower levels of body weight loss and disease activity index. Histologically, epithelial cell injury and inflammation were milder in the colonic mucosa of Spred2 KO mice on day 3 and almost undetectable by day 8. Experiments with bone chimeric mice indicated that Spred2-deficiency in non-hematopoietic cells was responsible for the reduced sensitivity to DSS. Finally, Spred2 KO mice developed significantly fewer tumors in response to azoxymethane plus DSS. Taken together, our results demonstrate, for the first time, that Spred2 plays an important role in the regulation of colonic epithelial cell proliferation and inflammation by potentially down-regulating the activation of ERK. Thus, Spred2 may be a new therapeutic target for the treatment of UC.
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19
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Nurr1 promotes intestinal regeneration after ischemia/reperfusion injury by inhibiting the expression of p21 (Waf1/Cip1). J Mol Med (Berl) 2016; 95:83-95. [PMID: 27553040 DOI: 10.1007/s00109-016-1464-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Revised: 07/20/2016] [Accepted: 08/15/2016] [Indexed: 12/20/2022]
Abstract
Intestinal ischemia/reperfusion (I/R) injury is a potentially life-threatening condition that can cause injuries to remote organs at the end stage. The damage caused by intestinal I/R insult induces changes in the barrier functions of the intestine, and the intrinsic mechanism of regeneration is often insufficient to restore barrier functions, as indicated by the high mortality rate of patients experiencing intestinal I/R injury. However, little is known about the mechanisms of intestinal regeneration after I/R injury. Here, we reported that nuclear receptor-related protein 1 (Nurr1), a nuclear orphan receptor, was induced during intestinal regeneration after I/R. Our findings showed that Nurr1 expression was consistent with the expression of Ki-67 and phosphorylated histone H3 (pH 3) in the intestine after I/R injury. Nurr1 knockdown led to G1-phase arrest mediated by p21 (Waf1/Cip1) activation, but Nurr1 overexpression reduced the proportion of IEC-6 cells in G1 phase as a result of p21 inhibition in a p53-independent manner. Using chromatin immunoprecipitation assays, luciferase assays, and mutational analysis, we demonstrated that Nurr1 directly inhibited the transcription of p21. These results define a novel Nurr1/p21 pathway that is involved in intestinal regeneration after I/R injury. These findings provide novel molecular insights into the pathogenesis of intestinal regeneration after I/R and possibly support the development of new potential therapies for intestinal I/R injury. KEY MESSAGE Nurr1 was induced during intestinal regeneration after I/R injury. Nurr1 promoted proliferation of intestinal epithelial cells after H/R injury. Nurr1 inhibited p21 expression in a p53-independent manner. Nurr1 inhibited p21 gene transcription by binding to p21 promoter directly.
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20
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Rager TM, Olson JK, Zhou Y, Wang Y, Besner GE. Exosomes secreted from bone marrow-derived mesenchymal stem cells protect the intestines from experimental necrotizing enterocolitis. J Pediatr Surg 2016; 51:942-7. [PMID: 27015901 PMCID: PMC4921266 DOI: 10.1016/j.jpedsurg.2016.02.061] [Citation(s) in RCA: 111] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Accepted: 02/26/2016] [Indexed: 12/27/2022]
Abstract
PURPOSE Treatment options for necrotizing enterocolitis (NEC) remain inadequate. Bone marrow-derived mesenchymal stem cells (BM-MSCs) can protect the intestines from NEC. Exosomes are nanoparticle-sized vesicles with important cell signaling capabilities. The objective of this study was to determine whether BM-MSC-derived exosomes can prevent NEC. METHODS Rat pups were either breast fed (Group 1) or subjected to experimental NEC and randomized to receive either no treatment (Group 2) or an intraperitoneal (IP) injection of PBS (Group 3), BM-MSC (Group 4), or BM-MSC-derived exosomes (Group 5). Histologic injury grade and intestinal permeability were determined. The effect of BM-MSC-derived exosomes on IEC-6 intestinal epithelial cells in an in vitro scrape model of wound healing was also determined. RESULTS Animals exposed to NEC that were either untreated or received PBS alone had an NEC incidence of 46% and 41%, respectively (p=0.61). Compared to untreated pups, the incidence of NEC was significantly lower in pups treated with either BM-MSC (9%, p=0.0003) or MB-MSC-derived exosomes (13%, p=0.0008). Similar results were found for intestinal permeability. Wound healing in IEC-6 cells was significantly increased by BM-MSC-derived exosomes. CONCLUSION BM-MSC-derived exosomes protect the intestines from NEC and may represent a novel, cell-free, preventative therapy for NEC in the future.
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21
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Ralls MW, Demehri FR, Feng Y, Woods Ignatoski KM, Teitelbaum DH. Enteral nutrient deprivation in patients leads to a loss of intestinal epithelial barrier function. Surgery 2015; 157:732-42. [PMID: 25704423 DOI: 10.1016/j.surg.2014.12.004] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2013] [Revised: 11/17/2014] [Accepted: 12/03/2014] [Indexed: 12/12/2022]
Abstract
OBJECTIVE To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens. DESIGN Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients. RESULTS Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual. CONCLUSION EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.
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Affiliation(s)
- Matthew W Ralls
- Section of Pediatric Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI
| | - Farokh R Demehri
- Section of Pediatric Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI
| | - Yongjia Feng
- Section of Pediatric Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI
| | | | - Daniel H Teitelbaum
- Section of Pediatric Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI.
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Goel G, Conway KL, Jaeger M, Netea MG, Xavier RJ. Multivariate inference of pathway activity in host immunity and response to therapeutics. Nucleic Acids Res 2014; 42:10288-306. [PMID: 25147207 PMCID: PMC4176341 DOI: 10.1093/nar/gku722] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Developing a quantitative view of how biological pathways are regulated in response to environmental factors is central for understanding of disease phenotypes. We present a computational framework, named Multivariate Inference of Pathway Activity (MIPA), which quantifies degree of activity induced in a biological pathway by computing five distinct measures from transcriptomic profiles of its member genes. Statistical significance of inferred activity is examined using multiple independent self-contained tests followed by a competitive analysis. The method incorporates a new algorithm to identify a subset of genes that may regulate the extent of activity induced in a pathway. We present an in-depth evaluation of specificity, robustness, and reproducibility of our method. We benchmarked MIPA's false positive rate at less than 1%. Using transcriptomic profiles representing distinct physiological and disease states, we illustrate applicability of our method in (i) identifying gene–gene interactions in autophagy-dependent response to Salmonella infection, (ii) uncovering gene–environment interactions in host response to bacterial and viral pathogens and (iii) identifying driver genes and processes that contribute to wound healing and response to anti-TNFα therapy. We provide relevant experimental validation that corroborates the accuracy and advantage of our method.
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Affiliation(s)
- Gautam Goel
- Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Kara L Conway
- Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Martin Jaeger
- Department of Internal Medicine and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen 6525 GA, The Netherlands
| | - Mihai G Netea
- Department of Internal Medicine and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen 6525 GA, The Netherlands
| | - Ramnik J Xavier
- Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA 02142, USA
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Watkins DJ, Zhou Y, Matthews MAB, Chen L, Besner GE. HB-EGF augments the ability of mesenchymal stem cells to attenuate intestinal injury. J Pediatr Surg 2014; 49:938-44; discussion 944. [PMID: 24888839 PMCID: PMC4044538 DOI: 10.1016/j.jpedsurg.2014.01.030] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/16/2014] [Accepted: 01/27/2014] [Indexed: 12/15/2022]
Abstract
BACKGROUND We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) and mesenchymal stem cell (MSC) administration protect the intestines from ischemia/reperfusion (I/R) injury in vivo, with amniotic fluid-derived MSC (AF-MSC) being more efficacious than bone marrow-derived MSC (BM-MSC). The goal of the current study was to determine whether the protective effects of HB-EGF were from direct effects on MSC or via alternative mechanisms. METHODS Murine MSC were transfected with an HB-EGF plasmid or control plasmid by electroporation. Mice were subjected to segmental intestinal I/R injury and received either BM-MSC or AF-MSC either with or without exogenous HB-EGF, or BM-MSC or AF-MSC that endogenously over-expressed HB-EGF. MSC engraftment, intestinal histologic injury, and intestinal permeability were quantified. RESULTS There was increased MSC engraftment into injured compared to uninjured intestine. HB-EGF increased AF-MSC engraftment into injured intestine. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury, with AF-MSC being most efficacious. The effect of HB-EGF on MSC was similar when the growth factor was administered exogenously, or when it was overexpressed endogenously. CONCLUSIONS The effect of HB-EGF on AF-MSC was similar with both exogenous administration and endogenous overexpression of the growth factor, implying that HB-EGF has a direct effect on AF-MSC. This information may assist in guiding potential future AF-MSC-based therapies for patients at risk of intestinal ischemic injuries.
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Affiliation(s)
- Daniel J Watkins
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio
| | - Yu Zhou
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio
| | - Mika A B Matthews
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio
| | - Li Chen
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio
| | - Gail E Besner
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio.
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Su Y, Besner GE. Heparin-binding EGF-like growth factor (HB-EGF) promotes cell migration and adhesion via focal adhesion kinase. J Surg Res 2014; 189:222-31. [PMID: 24703506 DOI: 10.1016/j.jss.2014.02.055] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2013] [Revised: 12/29/2013] [Accepted: 02/27/2014] [Indexed: 11/16/2022]
Abstract
BACKGROUND Cell migration and adhesion are essential in intestinal epithelial wound healing and recovery from injury. Focal adhesion kinase (FAK) plays an important role in cell-extracellular matrix signal transduction. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) promotes intestinal epithelial cell (IEC) migration and adhesion in vitro. The present study was designed to determine whether FAK is involved in HB-EGF-induced IEC migration and adhesion. MATERIALS AND METHODS A scrape wound healing model of rat IECs was used to examine the effect of HB-EGF on FAK-dependent cell migration in vitro. Immunofluorescence and Western blot analyses were performed to evaluate the effect of HB-EGF on the expression of phosphorylated FAK (p-FAK). Cell adhesion assays were performed to determine the role of FAK in HB-EGF-induced cell adhesion on fibronectin (FN). RESULTS HB-EGF significantly increased healing after scrape wounding, an effect that was reversed in the presence of an FAK inhibitor 14 (both with P < 0.05). HB-EGF increased p-FAK expression and induced p-FAK redistribution and actin reorganization in migrating rat IECs. Cell adhesion and spreading on FN were significantly increased by HB-EGF (P < 0.05). FAK inhibitor 14 significantly inhibited both intrinsic and HB-EGF-induced cell adhesion and spreading on FN (both with P < 0.05). CONCLUSIONS FAK phosphorylation and FAK-mediated signal transduction play essential roles in HB-EGF-mediated IEC migration and adhesion.
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Affiliation(s)
- Yanwei Su
- Department of Cardiovascular and Respiratory Medicine, Wuhan Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Department of Pediatric Surgery, Nationwide Children's Hospital, Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, Ohio.
| | - Gail E Besner
- Department of Pediatric Surgery, Nationwide Children's Hospital, Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, Ohio.
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25
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Chen CL, Yang J, James IOA, Zhang HY, Besner GE. Heparin-binding epidermal growth factor-like growth factor restores Wnt/β-catenin signaling in intestinal stem cells exposed to ischemia/reperfusion injury. Surgery 2014; 155:1069-80. [PMID: 24856127 DOI: 10.1016/j.surg.2014.01.013] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2013] [Accepted: 01/31/2014] [Indexed: 12/29/2022]
Abstract
BACKGROUND We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) protects the intestines from injury in several different experimental animal models. In the current study, we investigated whether the ability of HB-EGF to protect the intestines from ischemia/reperfusion (I/R) injury was related to its effects on Wnt/β-catenin signaling in intestinal stem cells (ISC). METHODS Lucien-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-enhanced green fluorescent protein (EGFP) transgenic (TG) mice with fluorescently labeled ISC, as well as the same mice treated with intraluminal HB-EGF or genetically engineered to overexpress HB-EGF, were exposed to segmental mesenteric artery occlusion (sMAO) to the terminal ilium. Wnt/β-catenin signaling was evaluated using immunofluorescent staining and Western blotting. RESULTS LGR5 expression and Wnt/β-catenin signaling in the ISC of the terminal ilium of LGR5-EGFP TG mice was significantly reduced 24 hours after sMAO. Intraluminal administration of HB-EGF or HB-EGF overexpression in these mice led to preservation of LGR5 expression and Wnt/β-catenin signaling. CONCLUSION These data show that HB-EGF preserves Wnt/β-catenin signaling in ISC after I/R injury.
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Affiliation(s)
- Chun-Liang Chen
- Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH; Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX
| | - Jixin Yang
- Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH
| | - Iyore O A James
- Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH
| | - Hong-Yi Zhang
- Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH
| | - Gail E Besner
- Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH.
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Yang J, Su Y, Zhou Y, Besner GE. Heparin-binding EGF-like growth factor (HB-EGF) therapy for intestinal injury: Application and future prospects. ACTA ACUST UNITED AC 2013; 21:95-104. [PMID: 24345808 DOI: 10.1016/j.pathophys.2013.11.008] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Throughout the past 20 years, we have been investigating the potential therapeutic roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor family, in various models of intestinal injury including necrotizing enterocolitis (NEC), intestinal ischemia/reperfusion (I/R) injury, and hemorrhagic shock and resuscitation (HS/R). Our studies have demonstrated that HB-EGF acts as an effective mitogen, a restitution-inducing reagent, a cellular trophic factor, an anti-apoptotic protein and a vasodilator, via its effects on various cell types in the intestine. In the current paper, we have reviewed the application and therapeutic effects of HB-EGF in three classic animal models of intestinal injury, with particular emphasis on its protection of the intestines from NEC. Additionally, we have summarized the protective functions of HB-EGF on various target cells in the intestine. Lastly, we have provided a brief discussion focusing on the future development of HB-EGF clinical applications for the treatment of various forms of intestinal injury including NEC.
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Affiliation(s)
- Jixin Yang
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH 43205, USA.
| | - Yanwei Su
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH 43205, USA.
| | - Yu Zhou
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH 43205, USA.
| | - Gail E Besner
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH 43205, USA.
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27
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Jiang H, Qu L, Dou R, Lu L, Bian S, Zhu W. Potential role of mesenchymal stem cells in alleviating intestinal ischemia/reperfusion impairment. PLoS One 2013; 8:e74468. [PMID: 24058571 PMCID: PMC3772852 DOI: 10.1371/journal.pone.0074468] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2013] [Accepted: 07/31/2013] [Indexed: 01/01/2023] Open
Abstract
Background Transplantation of bone marrow mesenchymal stem cells (MSCs) provides a promising therapeutic efficiency for a variety of disorders caused by ischemia or reperfusion impairment. We have previously demonstrated the efficacy of MSCs in mitigating intestinal ischemia/reperfusion (I/R) injuries in rats, but the mechanism by which MSCs engraft ameliorates I/R injuries has largely been unknown. The present study aimed at investigating probable mechanisms by which MSCs exert their function. Methods Male donor derived rat MSCs were implanted into intestine of female recipient rat by direct submucosal injection after superior mesenteric artery clamping and unclamping. The homed MSCs were detected by Y chromosome insitu hybridization probe, and the tumor necrosis factor-α (TNF-α) content in intestinal mucosa was determined by ELISA. Expression of proliferative cell nuclear antigen (PCNA) in bowel mucosa was assayed by real-time PCR and intestinal mucosa expression of phosphorylation extracellular signal-regulated kinase (pERK1/2) and nuclear factor-κB (NF-κB) were evaluated by western blot. Results Four and seven days after MSCs transplantation, the TNF-α content of bowel mucosa in MSCs group was significantly lower than that in saline group. The PCNA in bowel mucosa showed higher expression in MSCs treated group than the saline group, both at 4 and 7 days after cell transplantation. The expression of intestinal mucosal pERK1/2 in MSCs treated group was markedly higher than that in saline group, and the expression of NF-κB in MSCs treated group was noticeably decreased than that in saline group at 4 and 7 days post MSCs transplantation. Conclusion The present investigation provides novel evidence that MSCs have the potential to reduce intestinal I/R injuries probably due to their ability to accelerate cell proliferation and decrease the inflammatory response within intestinal mucosa after ischemia and reperfusion.
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Affiliation(s)
- Haitao Jiang
- Department of General Surgery Ⅱ， Affiliated Hospital of Qingdao University Medical College, Qingdao, China
- * E-mail:
| | - Linlin Qu
- Department of General Surgery Ⅱ， Affiliated Hospital of Qingdao University Medical College, Qingdao, China
| | - Rongrong Dou
- Department of General Surgery Ⅱ， Affiliated Hospital of Qingdao University Medical College, Qingdao, China
| | - Lianfang Lu
- Department of General Surgery Ⅱ， Affiliated Hospital of Qingdao University Medical College, Qingdao, China
| | - Sishan Bian
- Department of General Surgery, Shandong Provincial Hospital of Traditional Chinese Medicine, Jinan, China
| | - Weiming Zhu
- Research Institute of General Surgery, Jinling Hospital, Nanjing University, Nanjing, China
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Heparin-binding epidermal growth factor-like growth factor attenuates acute lung injury and multiorgan dysfunction after scald burn. J Surg Res 2013; 185:329-37. [PMID: 23777985 DOI: 10.1016/j.jss.2013.05.064] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2012] [Revised: 04/09/2013] [Accepted: 05/15/2013] [Indexed: 01/09/2023]
Abstract
BACKGROUND Impaired gut barrier function and acute lung injury (ALI) are significant components of the multiorgan dysfunction syndrome that accompanies severe burns. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to reduce inflammation, preserve gut barrier function, and protect the lungs from acute injury in several models of intestinal injury; however, comparable effects of HB-EGF after burn injury have never been investigated. The present studies were based on the hypothesis that HB-EGF would reduce the severity of ALI and multiorgan dysfunction after scald burns in mice. MATERIALS AND METHODS Mice were randomized to sham, burn (25% of total body surface area with full thickness dorsal scald), and burn + HB-EGF groups. The HB-EGF group was pretreated with two enteral doses of HB-EGF (1200 μg/kg/dose). Mice were resuscitated after injury and sacrificed at 8 h later. Their lungs were harvested for determination of pulmonary myeloperoxidase activity, wet:dry ratios, and terminal deoxynucleotidyl transferase dUTP nick end label and cleaved caspase 3 immunohistochemistry. Lung function was assessed using the SCIREQ Flexivent. Splenic apoptosis was quantified by Western blot for cleaved caspase 3, and intestinal permeability was measured using the everted gut sac method. RESULTS Mice subjected to scald burn injury had increased lung myeloperoxidase levels, increased pulmonary and splenic apoptosis, elevated airway resistance and bronchial reactivity, and increased intestinal permeability compared with sham mice. These abnormalities were significantly attenuated in mice that were subjected to scald burn injury but treated with enteral HB-EGF. CONCLUSIONS These data suggest that HB-EGF protects mice from ALI after scald burn and attenuates the severity of postburn multiorgan dysfunction.
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Watkins DJ, Yang J, Matthews MAB, Besner GE. Synergistic effects of HB-EGF and mesenchymal stem cells in a murine model of intestinal ischemia/reperfusion injury. J Pediatr Surg 2013; 48:1323-9. [PMID: 23845626 PMCID: PMC3710437 DOI: 10.1016/j.jpedsurg.2013.03.032] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/19/2013] [Accepted: 03/08/2013] [Indexed: 12/29/2022]
Abstract
BACKGROUND We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) administration protects the intestines from ischemia/reperfusion (I/R) injury in vivo. We have also shown that HB-EGF promotes mesenchymal stem cell (MSC) proliferation and migration in vitro. The goals of the current study were to examine the effects of HB-EGF and both bone marrow (BM)- and amniotic fluid (AF)-derived MSC on intestinal I/R injury in vivo. MATERIALS AND METHODS MSC were isolated from pan-EGFP mice, expanded, and purified. Pluripotency was confirmed by induced differentiation. Mice were subjected to terminal ileum I/R and received either: (1) no therapy; (2) HB-EGF; (3) BM-MSC; (4) HB-EGF+BM-MSC; (5) AF-MSC; or (6) HB-EGF+AF-MSC. MSC engraftment, histologic injury, and intestinal permeability were quantified. RESULTS There was increased MSC engraftment into injured compared to uninjured intestine for all experimental groups, with significantly increased engraftment for AF-MSC+HB-EGF compared to AF-MSC alone. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury. The greatest improvement was with combined administration of HB-EGF+AF-MSC. CONCLUSIONS Both HB-EGF alone and MSC alone can protect the intestines from I/R injury, with synergistic efficacy occurring when HB-EGF and AF-MSC are administered together.
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Affiliation(s)
- Daniel J Watkins
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Columbus, OH 43205, USA
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Innate immune signaling in the pathogenesis of necrotizing enterocolitis. Clin Dev Immunol 2013; 2013:475415. [PMID: 23762089 PMCID: PMC3677005 DOI: 10.1155/2013/475415] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2013] [Revised: 05/01/2013] [Accepted: 05/07/2013] [Indexed: 01/01/2023]
Abstract
Necrotizing enterocolitis (NEC) is a challenging disease to treat, and caring for patients afflicted by it remains both frustrating and difficult. While NEC may develop quickly and without warning, it may also develop slowly, insidiously, and appear to take the caregiver by surprise. In seeking to understand the molecular and cellular processes that lead to NEC development, we have identified a critical role for the receptor for bacterial lipopolysaccharide (LPS) toll like receptor 4 (TLR4) in the pathogenesis of NEC, as its activation within the intestinal epithelium of the premature infant leads to mucosal injury and reduced epithelial repair. The expression and function of TLR4 were found to be particularly elevated within the intestinal mucosa of the premature as compared with the full-term infant, predisposing to NEC development. Importantly, factors within both the enterocyte itself, such as heat shock protein 70 (Hsp70), and in the extracellular environment, such as amniotic fluid, can curtail the extent of TLR4 signaling and reduce the propensity for NEC development. This review will highlight the critical TLR4-mediated steps that lead to NEC development, with a focus on the proinflammatory responses of TLR4 signaling that have such devastating consequences in the premature host.
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31
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Rowland KJ, Choi PM, Warner BW. The role of growth factors in intestinal regeneration and repair in necrotizing enterocolitis. Semin Pediatr Surg 2013; 22:101-11. [PMID: 23611614 PMCID: PMC3635039 DOI: 10.1053/j.sempedsurg.2013.01.007] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Necrotizing enterocolitis (NEC) is a devastating intestinal disease resulting in major neonatal morbidity and mortality. The pathology is poorly understood, and the means of preventing and treating NEC are limited. Several endogenous growth factors have been identified as having important roles in intestinal growth as well as aiding intestinal repair from injury or inflammation. In this review, we will discuss several growth factors as mediators of intestinal regeneration and repair as well as potential therapeutic agents for NEC.
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Affiliation(s)
| | | | - Brad W. Warner
- Correspondence: Brad W. Warner, M.D. St. Louis Children's Hospital One Children's Place; Suite 5S40 St. Louis MO 63110 (314) 454-6022 - Phone (314) 454-2442 – Fax
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32
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Turk HF, Monk JM, Fan YY, Callaway ES, Weeks B, Chapkin RS. Inhibitory effects of omega-3 fatty acids on injury-induced epidermal growth factor receptor transactivation contribute to delayed wound healing. Am J Physiol Cell Physiol 2013; 304:C905-17. [PMID: 23426968 DOI: 10.1152/ajpcell.00379.2012] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Epidermal growth factor receptor (EGFR)-mediated signaling is required for optimal intestinal wound healing. Since n-3 polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA), alter EGFR signaling and suppress downstream activation of key signaling pathways, we hypothesized that DHA would be detrimental to the process of intestinal wound healing. Using a mouse immortalized colonocyte model, DHA uniquely reduced EGFR ligand-induced receptor activation, whereas DHA and its metabolic precursor eicosapentaenoic acid (EPA) reduced wound-induced EGFR transactivation compared with control (no fatty acid or linoleic acid). Under wounding conditions, the suppression of EGFR activation was associated with a reduction in downstream activation of cytoskeletal remodeling proteins (PLCγ1, Rac1, and Cdc42). Subsequently, DHA and EPA reduced cell migration in response to wounding. Mice were fed a corn oil-, DHA-, or EPA-enriched diet prior to intestinal wounding (2.5% dextran sodium sulfate for 5 days followed by termination after 0, 3, or 6 days of recovery). Mortality was increased in EPA-fed mice and colonic histological injury scores were increased in EPA- and DHA-fed mice compared with corn oil-fed (control) mice. Although kinetics of colonic EGFR activation and downstream signaling (PLCγ1, Rac1, and Cdc42) were delayed by both n-3 PUFA, colonic repair was increased in EPA- relative to DHA-fed mice. These results indicate that, during the early response to intestinal wounding, DHA and EPA uniquely delay the activation of key wound-healing processes in the colon. This effect is mediated, at least in part, via suppression of EGFR-mediated signaling and downstream cytoskeletal remodeling.
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Affiliation(s)
- Harmony F Turk
- Program in Integrative Nutrition and Complex Diseases, Texas A & M University, College Station, TX 77843, USA
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Su Y, Yang J, Besner GE. HB-EGF promotes intestinal restitution by affecting integrin-extracellular matrix interactions and intercellular adhesions. Growth Factors 2013; 31:39-55. [PMID: 23305395 DOI: 10.3109/08977194.2012.755966] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Restitution is a critical form of intestinal epithelial cell (IEC) healing. We have previously shown that heparin-binding epidermal-like growth factor (HB-EGF) is necessary for IEC restitution; however, the mechanisms by which HB-EGF promotes restitution remain poorly understood. This study was designed to investigate whether HB-EGF promotes intestinal restitution by affecting integrin-extracellular matrix (ECM) interactions and intercellular adhesions. The effect of HB-EGF administration was examined in a murine necrotizing enterocolitis (NEC) model in vivo and an IEC line scrape-wound healing model in vitro. We evaluated the effect of HB-EGF on the expression of integrins, E-cadherin/β-catenin, and integrin α5β1-dependent cell-ECM interactions. We found that HB-EGF promoted intestinal restitution and the expression of integrin α5β1. HB-EGF promoted integrin α5β1-dependent cell adhesion and spreading. In addition, HB-EGF decreased the expression E-cadherin/β-catenin, via the activation of v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB-1). We conclude that HB-EGF promotes intestinal restitution by affecting integrin-ECM interactions and intercellular adhesions.
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Affiliation(s)
- Yanwei Su
- Department of Pediatric Surgery, Nationwide Children's Hospital, The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Ohio State University College of Medicine, Columbus, Ohio 43205, USA
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Yang J, Watkins D, Chen CL, Bhushan B, Zhou Y, Besner GE. Heparin-binding epidermal growth factor-like growth factor and mesenchymal stem cells act synergistically to prevent experimental necrotizing enterocolitis. J Am Coll Surg 2012; 215:534-45. [PMID: 22819639 DOI: 10.1016/j.jamcollsurg.2012.05.037] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Revised: 05/30/2012] [Accepted: 05/30/2012] [Indexed: 02/07/2023]
Abstract
BACKGROUND We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo. STUDY DESIGN In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined. RESULTS Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05). CONCLUSIONS Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.
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Affiliation(s)
- Jixin Yang
- The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and the Ohio State University College of Medicine, Columbus, OH, USA
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Yang J, Radulescu A, Chen CL, Zhang HY, James IO, Besner GE. Heparin-binding epidermal growth factor-like growth factor improves intestinal barrier function and reduces mortality in a murine model of peritonitis. Surgery 2012; 153:52-62. [PMID: 22703966 DOI: 10.1016/j.surg.2012.04.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2011] [Accepted: 04/12/2012] [Indexed: 11/29/2022]
Abstract
BACKGROUND The morbidity and mortality associated with bacterial peritonitis remain high. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent. The aim of this study was to evaluate the effect of HB-EGF in a model of murine peritonitis. METHODS HB-EGF(-/-) knockout (KO) mice and their HB-EGF(+/+) wild-type (WT) counterparts were subjected to sham operation, cecal ligation and puncture (CLP), or CLP with HB-EGF treatment (800 μg/kg IP daily). Villous length, intestinal permeability, intestinal epithelial cell (IEC) apoptosis, bacterial load in peritoneal fluid (PF) and mesenteric lymph nodes (MLN), inflammatory cytokine levels, and survival were determined. RESULTS After exposure to CLP, HB-EGF KO mice had significantly shorter villi (1.37 ± 0.13 vs 1.96 ± 0.4 relative units; P < .03), increased intestinal permeability (17.01 ± 5.18 vs 11.50 ± 4.67 nL/min/cm2; P < .03), increased IEC apoptotic indices (0.0093 ± 0.0033 vs 0.0016 ± 0.0014; P < .01), and increased bacterial counts in PF (25,313 ± 17,558 vs 11,955 ± 6,653 colony forming units [CFU]/mL; P < .05) and MLN (19,009 ± 11,200 vs 5,948 ± 2,988 CFU/mL/g; P < .01) compared with WT mice. Administration of HB-EGF to WT and HB-EGF KO mice exposed to CLP led to significantly increased villous length and decreased intestinal permeability, IEC apoptosis and bacterial counts in MLN (P < .05). Survival of HB-EGF KO mice subjected to CLP was significantly improved with administration of HB-EGF (P < .05). CONCLUSION HB-EGF gene KO increases susceptibility to peritonitis-induced intestinal injury, which can be reversed by administration of HB-EGF. These results support a protective role of HB-EGF in peritonitis-induced sepsis.
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Affiliation(s)
- Jixin Yang
- Department of Pediatric Surgery, Nationwide Children's Hospital, Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, OH, USA
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Watkins DJ, Zhou Y, Chen CL, Darbyshire A, Besner GE. Heparin-binding epidermal growth factor-like growth factor protects mesenchymal stem cells. J Surg Res 2012; 177:359-64. [PMID: 22658491 DOI: 10.1016/j.jss.2012.05.016] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2012] [Revised: 04/23/2012] [Accepted: 05/03/2012] [Indexed: 01/09/2023]
Abstract
BACKGROUND We have previously demonstrated that mesenchymal stem cell (MSC) administration protects the intestines from injury in a mouse model of intestinal ischemia/reperfusion injury. We have also shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent in vivo that can protect the intestines by way of its effects on stem cells. The goal of the present study was to examine the effects of HB-EGF on both amniotic fluid (AF)- and bone marrow (BM)-derived MSCs in vitro. METHODS MSCs were isolated from the AF and BM of pan-EGFP mice, grown in MSC-specific culture medium, and purified by sequential passages according to their adherence properties. Pluripotency was confirmed by induced differentiation. After incubation of MSCs with HB-EGF, proliferation was quantified using the CyQuant cell proliferation assay kit under normoxic and anoxic conditions. Chemotaxis was quantified using the CHEMICON QCM cell migration kit, and apoptosis was determined using caspase-3 immunohistochemistry after exposure of the MSCs to anoxic stress. RESULTS AF-MSCs and BM-MSCs showed significantly increased proliferation and migration in response to HB-EGF. HB-EGF significantly protected AF-MSCs and BM-MSCs from anoxia-induced apoptosis. The proliferative and anti-apoptotic effects of HB-EGF were even more pronounced in AF-MSCs than in BM-MSCs. CONCLUSIONS These results have demonstrated that HB-EGF acts as a mitogenic and chemotactic agent for MSCs that protects MSCs from injury. These findings could have important implications for future experiments designed to use MSCs to protect the intestines from injury.
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Affiliation(s)
- Daniel J Watkins
- Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, Center for Perinatal Research, Ohio State University College of Medicine, Columbus, Ohio, USA
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Chen CL, Yu X, James IOA, Zhang HY, Yang J, Radulescu A, Zhou Y, Besner GE. Heparin-binding EGF-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing enterocolitis. J Transl Med 2012; 92:331-44. [PMID: 22157721 PMCID: PMC3289750 DOI: 10.1038/labinvest.2011.167] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.
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Zhang HY, James I, Chen CL, Besner GE. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) preserves gut barrier function by blocking neutrophil-endothelial cell adhesion after hemorrhagic shock and resuscitation in mice. Surgery 2011; 151:594-605. [PMID: 22153812 DOI: 10.1016/j.surg.2011.10.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2011] [Accepted: 10/07/2011] [Indexed: 11/17/2022]
Abstract
BACKGROUND We have shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) protects the intestines from injury in several different animal models, including hemorrhagic shock and resuscitation (HS/R). The current study was designed to explore the mechanisms underlying the anti-inflammatory role of HB-EGF in preservation of gut barrier function after injury. METHODS In vivo, HS/R was induced in wild-type and neutropenic mice, with or without administration of HB-EGF, and intestinal permeability determined by use of the everted gut sac method. In vitro, cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated human peripheral blood mononuclear cells (PMNs) were used to determine the effects of HB-EGF on HUVEC-PMN adhesion, reactive oxygen species production in PMN, adhesion molecule expression in HUVEC and PMN, and the signaling pathways involved. RESULTS We found that administration of HB-EGF to healthy mice led to preservation of gut barrier function after HS/R. Likewise, induction of neutropenia in mice also led to preservation of gut barrier function after HS/R. Administration of HB-EGF to neutropenic mice did not lead to further improvement in gut barrier function. In vitro studies showed that HB-EGF decreased neutrophil-endothelial cell (PMN-EC) adherence by down-regulating adhesion molecule expression in EC via the phosphoinositide 3-kinase-Akt pathway, and by inhibiting adhesion molecule surface mobilization and reactive oxygen species production in PMN. CONCLUSION These results indicate that HB-EGF preserves gut barrier function by inhibiting PMN and EC activation, thereby blocking PMN-EC adherence after HS/R in mice, and support the future use of HB-EGF in disease states manifested by hypoperfusion injury.
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Affiliation(s)
- Hong-yi Zhang
- Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA
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Zheng X, Tsuchiya K, Okamoto R, Iwasaki M, Kano Y, Sakamoto N, Nakamura T, Watanabe M. Suppression of hath1 gene expression directly regulated by hes1 via notch signaling is associated with goblet cell depletion in ulcerative colitis. Inflamm Bowel Dis 2011; 17:2251-60. [PMID: 21987298 DOI: 10.1002/ibd.21611] [Citation(s) in RCA: 113] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/09/2010] [Accepted: 11/15/2010] [Indexed: 12/11/2022]
Abstract
BACKGROUND The transcription factor Atoh1/Hath1 plays crucial roles in the differentiation program of human intestinal epithelium cells (IECs). Although previous studies have indicated that the Notch signal suppresses the differentiation program of IEC, the mechanism by which it does so remains unknown. This study shows that the undifferentiated state is maintained by the suppression of the Hath1 gene in human intestine. METHODS To assess the effect of Notch signaling, doxycycline-induced expression of Notch intracellular domain (NICD) and Hes1 cells were generated in LS174T. Hath1 gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Hath1 promoter region targeted by HES1 was determined by both reporter analysis and ChIP assay. Expression of Hath1 protein in ulcerative colitis (UC) was examined by immunohistochemistry. RESULTS Hath1 mRNA expression was increased by Notch signal inhibition. However, Hath1 expression was suppressed by ectopic HES1 expression alone even under Notch signal inhibition. Suppression of the Hath1 gene by Hes1, which binds to the 5' promoter region of Hath1, resulted in suppression of the phenotypic gene expression for goblet cells. In UC, the cooperation of aberrant expression of HES1 and the disappearance of caudal type homeobox 2 (CDX2) caused Hath1 suppression, resulting in goblet cell depletion. CONCLUSIONS The present study suggests that Hes1 is essential for Hath1 gene suppression via Notch signaling. Moreover, the suppression of Hath1 is associated with goblet cell depletion in UC. Understanding the regulation of goblet cell depletion may lead to the development of new therapy for UC.
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Affiliation(s)
- Xiu Zheng
- Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
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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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Yamaoka T, Frey MR, Dise RS, Bernard JK, Polk DB. Specific epidermal growth factor receptor autophosphorylation sites promote mouse colon epithelial cell chemotaxis and restitution. Am J Physiol Gastrointest Liver Physiol 2011; 301:G368-76. [PMID: 21617115 PMCID: PMC3154598 DOI: 10.1152/ajpgi.00327.2010] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Upon ligand binding, epidermal growth factor (EGF) receptor (R) autophosphorylates on COOH-terminal tyrosines, generating docking sites for signaling partners that stimulate proliferation, restitution, and chemotaxis. Specificity for individual EGFR tyrosines in cellular responses has been hypothesized but not well documented. Here we tested the requirement for particular tyrosines, and associated downstream pathways, in mouse colon epithelial cell chemotactic migration. We compared these requirements to those for the phenotypically distinct restitution (wound healing) migration. Wild-type, Y992/1173F, Y1045F, Y1068F, and Y1086F EGFR constructs were expressed in EGFR(-/-) cells; EGF-induced chemotaxis or restitution were determined by Boyden chamber or modified scratch wound assay, respectively. Pharmacological inhibitors of p38, phospholipase C (PLC), Src, MEK, JNK/SAPK, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC) were used to block EGF-stimulated signaling. Pathway activation was determined by immunoblot analysis. Unlike wild-type EGFR, Y992/1173F and Y1086F EGFR did not stimulate colon epithelial cell chemotaxis toward EGF; Y1045F and Y1068F EGFR partially stimulated chemotaxis. Only wild-type EGFR promoted colonocyte restitution. Inhibition of p38, PLC, and Src, or Grb2 knockdown, blocked chemotaxis; JNK, PI 3-kinase, and PKC inhibitors or c-Cbl knockdown blocked restitution but not chemotaxis. All four EGFR mutants stimulated downstream signaling in response to EGF, but Y992/1173F EGFR was partially defective in PLCγ activation whereas both Y1068F and Y1086F EGFR failed to activate Src. We conclude that specific EGFR tyrosines play key roles in determining cellular responses to ligand. Chemotaxis and restitution, which have different migration phenotypes and physiological consequences, have overlapping but not identical EGFR signaling requirements.
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Affiliation(s)
- Toshimitsu Yamaoka
- 1Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition and
| | - Mark R. Frey
- 3Department of Pediatrics, Division of Gastroenterology and Nutrition and ,4Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine and The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California
| | - Rebecca S. Dise
- 2Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee; and
| | - Jessica K. Bernard
- 3Department of Pediatrics, Division of Gastroenterology and Nutrition and
| | - D. Brent Polk
- 1Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition and ,2Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee; and ,3Department of Pediatrics, Division of Gastroenterology and Nutrition and ,4Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine and The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California
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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: 197] [Impact Index Per Article: 14.1] [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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Zhang HY, Radulescu A, Chen CL, Olson JK, Darbyshire AK, Besner GE. Mice overexpressing the gene for heparin-binding epidermal growth factor-like growth factor (HB-EGF) have increased resistance to hemorrhagic shock and resuscitation. Surgery 2010; 149:276-83. [PMID: 20965535 DOI: 10.1016/j.surg.2010.08.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2010] [Accepted: 08/05/2010] [Indexed: 12/28/2022]
Abstract
BACKGROUND The aim of the current study was to determine whether overexpression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) could protect the intestines from injury after hemorrhagic shock and resuscitation in mice. METHODS Hemorrhagic shock and resuscitation was induced in HB-EGF transgenic and wild type mice. Cross-reacting material 197 (5 mg/kg) was administered to a subset of HB-EGF transgenic mice to block the overexpressed HB-EGF. Intestinal histologic injury scores, intestinal epithelial cell apoptosis indices, and gut barrier function were determined. The Student t test and 1-way analysis of variance were employed to compare the differences between groups. RESULTS All mice subjected to hemorrhagic shock and resuscitation had significantly increased intestinal histologic injury scores, apoptosis indices, and intestinal permeability compared with sham-operated mice. Compared with wild type mice, HB-EGF transgenic mice had significantly decreased histologic injury (mean injury grade 2.79 ± 0.84 vs 3.88 ± 1.43, P = .02), apoptosis indices (mean apoptosis index 8.77 ± 5.23 vs 17.91 ± 13.23, P = .03), and mucosal permeability (FITC-dextran 4 clearance 13.06 ± 5.67 vs 20.03 ± 7.81 nL/min/ m(2), P = .02) at 3 hours of reperfusion. HB-EGF transgenic mice subjected to hemorrhagic shock and resuscitation and treated with cross-reacting material 197 had a significantly increased histologic injury (mean injury grade 3.63 ± 1.00 vs 2.79 ± 0.84, P = .04) and mucosal permeability (FITC-dextran 4 clearance 22.87 ± 9.69 vs 13.06 ± 5.67 nL/min/cm2, P = .01) at 3 hours of reperfusion compared with non-cross-reacting material 197 treated transgenic mice, with no significant changes in apoptosis indices. Cross-reacting material 197 did not reverse the decreased apoptosis observed in HB-EGF transgenic mice subjected to hemorrhagic shock and resuscitation, which suggests that mechanisms in addition to decreased apoptosis may be responsible for the intestinal cytoprotective effects of endogenous HB-EGF overexpression. CONCLUSION Overexpression of HB-EGF increases resistance to hemorrhagic shock and resuscitation in mice.
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Affiliation(s)
- Hong-yi Zhang
- Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Department of Pediatric Surgery, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH, USA
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Radulescu A, Zhang HY, Yu X, Olson JK, Darbyshire AK, Chen Y, Besner GE. Heparin-binding epidermal growth factor-like growth factor overexpression in transgenic mice increases resistance to necrotizing enterocolitis. J Pediatr Surg 2010; 45:1933-9. [PMID: 20920709 PMCID: PMC2953427 DOI: 10.1016/j.jpedsurg.2010.05.002] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2010] [Revised: 04/07/2010] [Accepted: 05/02/2010] [Indexed: 11/17/2022]
Abstract
BACKGROUND Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency and the leading surgical cause of death in premature infants. We have shown that administration of exogenous heparin-binding epidermal growth factor-like growth factor (HB-EGF) in mice protects the intestines from experimental NEC. The aim of the current study was to evaluate the effect of gain-of-function of endogenous HB-EGF on susceptibility to NEC. METHODS Neonatal HB-EGF transgenic (TG) mice and their wild-type (WT) counterparts were exposed to experimental NEC. An additional group of HB-EGF TG pups were also exposed to NEC, but received the HB-EGF antagonist cross-reacting material 197 (CRM197) injected subcutaneously immediately after birth. To examine gut barrier function, HB-EGF TG and WT pups received intragastric fluorescein isothiocyanate-labeled dextran under basal and stressed conditions, and serum fluorescein isothiocyanate-labeled dextran levels were measured. RESULTS Wild-type mice had an incidence of NEC of 54.2%, whereas HB-EGF TG mice had a significantly decreased incidence of NEC of 22.7% (P = .03). Importantly, administration of CRM197 to HB-EGF TG pups significantly increased the incidence of NEC to 65% (P = .004). HB-EGF TG mice had significantly decreased intestinal permeability compared to WT mice both under basal and stressed conditions. CONCLUSIONS Our results provide evidence that overexpression of the HB-EGF gene decreases susceptibility to NEC and that administration of the HB-EGF antagonist CRM197 reverses this protective effect.
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Affiliation(s)
- Andrei Radulescu
- Department of Pediatric Surgery, Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH 43205, USA
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Radulescu A, Zhang HY, Chen CL, Chen Y, Zhou Y, Yu X, Otabor I, Olson JK, Besner GE. Heparin-binding EGF-like growth factor promotes intestinal anastomotic healing. J Surg Res 2010; 171:540-50. [PMID: 20850767 DOI: 10.1016/j.jss.2010.06.036] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2010] [Revised: 06/15/2010] [Accepted: 06/24/2010] [Indexed: 11/29/2022]
Abstract
BACKGROUND We have accumulated multiple lines of evidence supporting the ability of HB-EGF to protect the intestines from injury and to augment the healing of partial-thickness scald burns of the skin. The aim of the current study was to investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in intestinal anastomotic wound healing. MATERIALS AND METHODS HB-EGF (-/-) knockout (KO) mice (n=42) and their HB-EGF (+/+) wild type (WT) counterparts (n=33), as well as HB-EGF transgenic (TG) mice (n=26) and their (WT) counterparts (n=27), underwent division and reanastomosis of the terminal ileum. In addition, WT mice (n=21) that received enteral HB-EGF (800 μg/kg) underwent the same operative procedure. Anastomotic bursting pressure was measured at 3 and 6 d postoperatively. Tissue sections were stained with hematoxylin and eosin to assess anastomotic healing, and Picrosirus red to assess collagen deposition. Immunohistochemistry using anti-von Willebrand factor antibodies was performed to assess angiogenesis. Complications and mortality were also recorded. RESULTS HB-EGF KO mice had significantly lower bursting pressures, lower healing scores, higher mortality, and higher complication rates postoperatively compared with WT mice. Collagen deposition and angiogenesis were significantly decreased in KO mice compared with WT mice. Conversely, HB-EGF TG mice had increased anastomotic bursting pressure, higher healing scores, lower mortality, lower complication rates, increased collagen deposition, and increased angiogenesis postoperatively compared with WT mice. WT mice that received HB-EGF had increased bursting pressures compared with non-HB-EGF treated mice. CONCLUSION Our results demonstrate that HB-EGF is an important factor involved in the healing of intestinal anastomoses.
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Affiliation(s)
- Andrei Radulescu
- Center for Perinatal Research, Department of Pediatric Surgery, The Research Institute at Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio 43205, USA
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James IAO, Chen CL, Huang G, Zhang HY, Velten M, Besner GE. HB-EGF protects the lungs after intestinal ischemia/reperfusion injury. J Surg Res 2010; 163:86-95. [PMID: 20599214 DOI: 10.1016/j.jss.2010.03.062] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2009] [Revised: 03/11/2010] [Accepted: 03/29/2010] [Indexed: 01/09/2023]
Abstract
BACKGROUND Acute respiratory distress syndrome continues to be a major source of morbidity and mortality in critically-ill patients. Heparin binding EGF-like growth factor (HB-EGF) is a biologically active protein that acts as an intestinal cytoprotective agent. We have previously demonstrated that HB-EGF protects the intestines from injury in several different animal models of intestinal injury. In the current study, we investigated the ability of HB-EGF to protect the lungs from remote organ injury after intestinal ischemia/reperfusion (I/R). METHODS Mice were randomly assigned to one of the following groups: (1) sham-operated; (2) sham+HB-EGF (1200 microg/kg in 0.6 mL administered by intra-luminal injection at the jejuno-ileal junction immediately after identification of the superior mesenteric artery); (3) superior mesenteric artery occlusion for 45 min followed by reperfusion for 6 h (I/R); or (4) I/R+HB-EGF (1200 microg/kg in 0.6 mL) administered 15 min after vascular occlusion. The severity of acute lung injury was determined by histology, morphometric analysis and invasive pulmonary function testing. Animal survival was evaluated using Kaplan-Meier analysis. RESULTS Mice subjected to intestinal I/R injury showed histologic and functional evidence of acute lung injury and decreased survival compared with sham-operated animals. Compared with mice treated with HB-EGF (I/R+HB-EGF), the I/R group had more severe acute lung injury, and decreased survival. CONCLUSION Our results demonstrate that HB-EGF reduces the severity of acute lung injury after intestinal I/R in mice. These data demonstrate that HB-EGF may be a potential novel systemic anti-inflammatory agent for the prevention of the systemic inflammatory response syndrome (SIRS) after intestinal injury.
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Affiliation(s)
- Iyore A O James
- Department of Pediatric Surgery, Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, Ohio, USA
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Radulescu A, Yu X, Orvets ND, Chen Y, Zhang HY, Besner GE. Deletion of the heparin-binding epidermal growth factor-like growth factor gene increases susceptibility to necrotizing enterocolitis. J Pediatr Surg 2010; 45:729-34. [PMID: 20385279 PMCID: PMC2855155 DOI: 10.1016/j.jpedsurg.2009.06.035] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/12/2009] [Revised: 06/26/2009] [Accepted: 06/29/2009] [Indexed: 10/19/2022]
Abstract
BACKGROUND Necrotizing enterocolitis (NEC) is the leading surgical cause of death in premature infants. We have accumulated evidence supporting a role for heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) in protection of the intestines from NEC. The aim of the current study was to evaluate the effect of loss-of-function of endogenous HB-EGF on susceptibility to NEC. METHODS Neonatal HB-EGF((-/-)) knockout (KO) mice and their HB-EGF((+/+)) wild-type (WT) counterparts were exposed to experimental NEC. An additional group of HB-EGF KO pups were also exposed to NEC but had HB-EGF added to their formula. To examine gut barrier function, HB-EGF KO and WT pups received intragastric fluorescein isothiocyanate-labeled dextran (FITC dextran) under basal and stressed conditions, and serum FITC dextran levels were measured. RESULTS The WT mice had an incidence of NEC of 53%, whereas HB-EGF KO mice had a significantly increased incidence of NEC of 80% (P = .04). Importantly, administration of exogenous HB-EGF to HB-EGF KO pups significantly reduced the incidence of NEC to 45% (P = .04). Heparin-binding EGF KO mice had significantly increased intestinal permeability compared to WT mice under basal and stressed conditions. CONCLUSIONS Our results provide evidence that loss of the HB-EGF gene increases susceptibility to NEC and that administration of exogenous HB-EGF reverses this susceptibility.
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Chen CL, Mehta VB, Zhang HY, Wu D, Otabor I, Radulescu A, El-Assal ON, Feng J, Chen Y, Besner GE. Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes. Growth Factors 2010; 28:82-97. [PMID: 19939201 PMCID: PMC3821006 DOI: 10.3109/08977190903407365] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
PRIMARY OBJECTIVE Heparin-binding EGF-like growth factor (HB-EGF) protects the intestine from damage in animals. Future clinical trials of HB-EGF may involve administration of repeated doses of HB-EGF. Since HB-EGF activates EGF receptors which have been implicated in tumor development, we examined the effects of HB-EGF overexpression in the intestine. RESEARCH DESIGN We generated transgenic (TG) mice in which the human HB-EGF gene is driven by the villin promoter to overexpress HB-EGF along the crypt-villous axis from the duodenum to the colon. RESULTS HB-EGF TG mice have increased enterocyte proliferation balanced by increased enterocyte apoptosis. Despite prolonged overexpression of HB-EGF, no evidence of intestinal hyperplasia or tumor formation occurs. Although HB-EGF TG mice have no significant phenotypic alterations under basal conditions, they have increased resistance to intestinal injury. CONCLUSIONS Prolonged intestinal HB-EGF overexpression results in no significant phenotypic alterations under basal conditions, but confers protection against intestinal injury.
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Affiliation(s)
- Chun-Liang Chen
- Department of Pediatric Surgery, The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH, USA
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Luu Y, Junker W, Rachagani S, Das S, Batra SK, Heinrikson RL, Shekels LL, Ho SB. Human intestinal MUC17 mucin augments intestinal cell restitution and enhances healing of experimental colitis. Int J Biochem Cell Biol 2010; 42:996-1006. [PMID: 20211273 DOI: 10.1016/j.biocel.2010.03.001] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2009] [Revised: 02/13/2010] [Accepted: 03/01/2010] [Indexed: 12/19/2022]
Abstract
UNLABELLED The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and are thought to be cytoprotective. The extracellular regions of these mucins contain EGF-like Cys-rich segments (CRD1 and CRD2) connected by an intervening linker domain (L). The purpose of this study was to determine the functional activity of human MUC17 membrane-bound mucin. METHODS Endogenous MUC17 was inhibited in LS174T colon cells by stable transfection of a small hairpin RNA targeting MUC17 (LSsi cells). The effect of recombinant MUC17-CRD1-L-CRD2 protein on migration, apoptosis, and experimental colitis was determined. RESULTS Reduced MUC17 expression in LSsi cells was associated with visibly reduced cell aggregation, reduced cell-cell adherence, and reduced cell migration, but no change in tumorigenicity. LSsi cells also demonstrated a 3.7-fold increase in apoptosis rates compared with control cells following treatment with etoposide. Exposure of colonic cell lines to exogenous recombinant MUC17-CRD1-L-CRD2 protein significantly increased cell migration and inhibited apoptosis. As a marker of biologic activity, MUC17-CRD1-L-CRD2 proteins stimulate ERK phosphorylation in colonic cell lines; and inhibition of ERK phosphorylation reduced the anti-apoptosis and migratory effect of MUC17-CRD1-L-CRD2. Finally, mice treated with MUC17-CRD1-L-CRD2 protein given per rectum demonstrated accelerated healing in acetic acid and dextran sodium sulfate induced colitis in vivo. These data indicate that both native MUC17 and the exogenous recombinant cysteine-rich domain of MUC17 play a role in diverse cellular mechanisms related to cell restitution, and suggest a potential role for MUC17-CRD1-L-CRD2 recombinant protein in the treatment of mucosal inflammatory diseases.
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Affiliation(s)
- Ying Luu
- Department of Medicine, VA San Diego Healthcare System and University of California San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161, United States
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Desai LP, White SR, Waters CM. Cyclic mechanical stretch decreases cell migration by inhibiting phosphatidylinositol 3-kinase- and focal adhesion kinase-mediated JNK1 activation. J Biol Chem 2010; 285:4511-9. [PMID: 20018857 PMCID: PMC2836056 DOI: 10.1074/jbc.m109.084335] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2009] [Revised: 11/30/2009] [Indexed: 01/11/2023] Open
Abstract
Epithelial cell migration during wound healing requires coordinated signaling pathways that direct polarization of the leading and trailing ends of the cells, cytoskeletal organization, and remodeling of focal adhesions. These inherently mechanical processes are disrupted by cyclic stretch (CS), but the specific signaling molecules involved in this disruption are not well understood. In this study, we demonstrate that inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of a dominant-negative form of PI3K caused inhibition of airway epithelial cell wound closure. CS caused a sustained decrease in activation of PI3K and inhibited wound healing. Expression of constitutively active PI3K stimulated translocation of Tiam1 to the membrane, increased Rac1 activity, and increased wound healing of airway epithelial cells. Increased Rac1 activity resulted in increased phosphorylation of JNK1. PI3K activation was not regulated by association with focal adhesion kinase. Restoration of efficient cell migration during CS required coexpression of constitutively active PI3K, focal adhesion kinase, and JIP3.
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Affiliation(s)
| | - Steven R. White
- the Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois 60637
| | - Christopher M. Waters
- From the Departments of Physiology
- Medicine, and
- Biomedical Engineering, University of Tennessee Health Science Center, Memphis, Tennessee 38163 and
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