DOCSLIB.ORG

The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial Cells Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1318 The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial Cells BY KRISTINA HOLMQVIST ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2004 ! "#$"% & ' ( & & )' ' * & +, -' . / (', 0 1 2, !, -' 3 & 4' 5 ( ( 6 7/6 4( ( / ' , 5 , "#", %% , , 849 :";%%!;%%";# 5 ( ( & ' & & . & ; ( , -' ($ ( & && & ' , 5 ( ( ( & & ( . ' & ' ' ( . ' & *7/6+ & ( . ' & *6+ ' & , 4' 1 . ' ' < , ' & ' & 4' 6; 7/6;( ( ' , 4' . & ' ' 4; ' 6; 7/6; , -' . & ( & ( ;4, ' 4'; ( ( & ' *8/+ . 4; , 6 . 4' 52 .'' . ' ' ' ( & 4', 8/ ( .; 3%2 4' * 40 + 52 ( , -' 40; & 4' . & ""=% ' 7/63; ' ' ( )5/ ( 7/63;, ' & 395 4' < <; . ' 4' ( 52 ( & & 7/6 & 7/63;, 8 4' ' 63;" 7/63; ( ' & 52 ' & & ( .'' & & & . , 8/ . ' 40 & 4' ( & & ' & .' & .; 4' ' ( , - < ( ' ' . ' ' 4' ' ( ( ( ( < ( . ' 52 ( ( ( & & ( , 4' 4 52 5 ( ( 7/63; 63;" / ' 4 ( 4 & & 395 7/6 6 && ( - & 395 ! " #! "$ %&'! ! ()&%'*+ ! > 2 0 1 ! 8449 ;=!= 849 :";%%!;%%";# $ $$$ ;#:!# *' $?? ,<,? @ A $ $$$ ;#:!#+ To myself for being determined and never giving up no matter what. 1 This thesis is based on the following papers, which will be referred to by their Roman numerals: I Lingge Lu, Kristina Holmqvist, Michael Cross and MichaelWelsh. Role of the Src Homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation. Cell Growth Differ 2002;13:141-148. II Kristina Holmqvist, Michael Cross, Debbie Riley and Michael Welsh. The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells. Cell Signal 2003;15:171-179. III Kristina Holmqvist, Michael Cross, Robert Hägerkvist, Nader Rahimi and Michael Welsh. The Shb adaptor protein binds to tyrosine 1175 in the VEGFR-2 and regulates FAK activity, stress fiber formation and cellular migration. Submitted. Reprints were made with the permission of the publishers. 2 TABLE OF CONTENTS ABSTRACT LIST OF PUBLICATIONS 2 TABLE OF CONTENTS 3 ABBREVIATIONS 5 INTRODUCTION 7 Angiogenesis 7 Receptor tyrosine kinases 9 FGFR-1 signalling 10 VEGFR-2 signalling 11 Shb 13 FAK 16 Src 18 Cytoskeleton 19 RNA interference 21 AIMS 22 METHODOLOGY 23 Cells 23 Protein assays; Immunoprecipitation 23 Western blot analysis 24 Fusion proteins, peptides and pull-down experiments 24 Rac1 and Rap1 assay 25 Rho assay 25 DNA synthesis 25 Proliferation and Viability 26 siRNA 26 Physiological assays; Tube formation 27 Morphology assay 27 Actin staining 27 Migration 28 Statistical analysis 28 3 RESULTS AND DISCUSSION 30 Paper I; The role of Shb in endothelial cell proliferation and differentiation. 30 Shb is necessary for tubular morphogenesis. 31 Paper II; Shb causes Src-dependent cell spreading and activation of FAK. 32 Shb and the downstream signalling of FGFR-1. 33 Paper III; Shb interacts with tyrosine 1175 in the VEGFR-2. 34 Shb regulates FAK activity, stress fiber formation and migration in VEGFR-2. 36 CONCLUSIONS 39 FUTURE PERSPECTIVES 40 ACKNOWLEDGEMENTS 41 REFERENCES 44 ORIGINAL PAPER 52 4 ABBREVIATIONS bFGF basic fibroblast growth factor (FGF-2) BSA Bovine serum albumin CHO Chinese hamster ovary CADTK Calcium dependent protein tyrosine kinase (Pyk2) CAK Cell adhesion kinase (Pyk2) cDNA complementary deoxyribonucleic acid CSF Colony stimulating factor CSK c-Src tyrosine kinase dsRNA double stranded RNA ECL Enhanced chemiluminescence ECM Extra cellular matrix EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor FAK Focal adhesion kinase FAT Focal adhesion targeting FCS Fetal calf serum FGF-2 Fibroblast growth factor 2 (bFGF) FGFR Fibroblast growth factor receptor FRNK FAK related non kinase GAP GTPase activating proteins GDI Guanine nucleotide dissociation inhibitor GEF Guanine exchange factor GPCR G-protein coupled receptor GST Glutatione-S-transferase HIF Hypoxia inducible factor HRE Hypoxia response element HRP Horseradish peroxidase HSPG Heparan sulphate proteoglycans IB Immunoblotting IBE cell Immortomouse brain endothelial cell IFN-J Interferon J IL-2R Interleukin 2 receptor IP Immunoprecipitation IPTG Isopropyl-thiogalaktoside LAT Linker for activation of T cell LPA Lysophosphatidic acid MAPK Mitogen activating protein kinase MLCK Myosin light chain kinase MMP Matrix metalloprotease NGF Nerve growth factor PAE Porcine aortic endothelial 5 PBS Phosphate buffered saline PDGF Platelet-derived growth factor PlGF Placenta growth factor PRNK Pyk2 related non kinase PTB Phosphotyrosine binding RAFTK Related adhesion focal tyrosine kinase (Pyk2) Rho Ras homologous RISC RNA induced silencing complex RNAi RNA interference RTK Receptor tyrosine kinase SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SH2/3 Src homology 2/3 SI/II Site I/II siRNA short interfering RNA SMC Smooth muscle cell TCR T-cell receptor TBS Tris buffered saline TBS-T Tris buffered saline-tween 20 TIME Telomerase immortalised microvascular endothelial TNFD Tumour necrosis factor D ts temperature sensitive VEGF Vascular endothelial growth factor VPF Vascular permeability factor (VEGF) VRAP VEGFR-associated protein 6 INTRODUCTION Angiogenesis Angiogenesis is defined as the formation of new capillary blood vessels from pre-existing ones. This process involves several steps including: migration, proliferation and differentiation of endothelial cells into blood vessels (Folkman et al., 1996, Risau et al., 1997). Angiogenesis is initiated by binding of specific growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to their cell surface receptors. This leads to receptor activation, an increase in the intrinsic tyrosine kinase activity and phosphorylation of specific intracellular tyrosine residues. As a consequence, certain signalling molecules bind to the phosphorylated tyrosines, and this initiates activation of downstream signalling cascades, leading to transcriptional changes and ultimately biological responses. Tumour cells secrete angiogenic factors, such as FGF and VEGF, that stimulate angiogenesis, facilitating the expansion of the tumour. VEGF and FGF can act synergistically in inducing angiogenesis (Stavri et al., 1995). The endothelial cells will, as a result of receptor activation, secrete proteases such as matrix metalloproteases (MMP) and plasminogen activators, leading to digestion of the basement membrane. This allows the endothelial cells to invade the surrounding tissue, where they migrate and proliferate to create a sprout (Fig. 1). The sprout elongates and the endothelial cells differentiate to form a functional lumen. The endothelial cells, in the newly formed vessel, will eventually start to secrete platelet-derived growth factor (PDGF-BB), which recruits supporting mesenchymal cells, that will differentiate into pericytes. This is critical for the stability of the vessel (Hirschi et al., 1997). Development of abnormal capillaries in pericyte-deficient PDGF-B -/- embryos shows that pericytes regulate microvessel structure (Lindahl et al., 1997). Physiological angiogenesis is restricted to embryonic development, ovulation and wound healing and in these cases the process is characterised by strict regulation. 7 Fig.1 Tumour cells ECM Migrat ion/ Proliferat ion & different iat ion Prot eolysis Angiogenic factors e.g. FGF & VEGF Blood vessel Pericyt e/ Endothelial Cell Smoot h muscle cell Figure 1. The process of angiogenesis in a tumour environment. However, in pathological conditions, such as arthritis, growth of tumours and in the formation of metastasis, vessel formation is exaggerated and the vessels may be non-functional and lacking supporting pericytes. This leads to leakage from the vessel, explaining the oedematous nature of most tumours. Already in 1971, Folkman suggested that a tumour requires blood supply in order to grow beyond a few cubic millimeters and thereby predicted, if pathological angiogenesis is inhibited, there will be a reduction of tumour growth and metastasis (Folkman et al., 1972). Today, a wealth of data supports this theory (O’Reilly et al., 1997, Boehm et al., 1997). The angiogenic switch, is triggered as a result of a shift in the balance between angiogenesis stimulators, bFGF and VEGF, and endogenous inhibitors, e.g. angiostatin and endostatin (Hanahan et al., 1996). Angiogenesis will be initiated when there is a change in the balance between activators and inhibitors, e.g. a reduction of inhibitor concentrations, by loss of tumour suppressor genes, or an increase in the levels of stimulators, by induction of VEGF, caused by hypoxia. In the absence of inducers or when the inducers are present but suppressed by higher levels of angiogenesis inhibitors the switch is off. Weidner et al., 1991 were the first to reveal a direct relationship 8 between metastasis and angiogenesis, in which they showed that a higher degree of angiogenesis in a primary tumour correlates with poor prognosis. Receptor tyrosine kinases Receptor tyrosine kinases (RTK) consist of an extracellular ligand binding domain, a transmembrane region and an intracellular
Load more

Recommended publications
  • Foundations of Biomedical Sciences Module IV
    Foundations in Biomedical Sciences IV: Mechanisms of Cell Communication Spring 2012 Tuesdays, Thursdays, and Fridays, 9:30 am to 11:20 am Fridays, 12:15 pm to 1:45 pm Course description The fourth module of the Foundations in Biomedical Sciences course will focus on the mechanisms of cell communication. This module will begin by discussing overarching concepts before examining the specific types of molecules that initiate and transduce signals. Examples of cell signaling and subsequent cellular responses will then be considered in different contexts to provide a framework on which future learning can be applied. As the module progresses, the complexity of the systems explored will increase from individual cells to multicellular environments such as tissues, organs and organisms. In addition, normal processes as well as the dysregulation of cell-cell communication in disease will be studied. The course will be aimed towards first year Ph.D. students in the Division of Graduate Medical Sciences. The class will be taught by members of the Division in a variety of Departments utilizing a combination of traditional lectures and discussion sections focusing on primary research. There will be a total of 7 hours of class time per week. Supplementary study materials will be made available using Blackboard (www.blackboard.bu.edu) to aid students in the review of the material. Reading materials will be taken primarily from the scientific literature and will utilize examples of classical studies as well as recent works. Students will be evaluated on their performance on a quiz, problem set, and examination along with active participation in discussion sections.
    [Show full text]
  • MINAR Is a Novel NOTCH-2 Interacting Protein That Regulates NOTCH-2 Activation and Angiogenesis
    Boston University OpenBU http://open.bu.edu Theses & Dissertations Boston University Theses & Dissertations 2017 MINAR is a novel NOTCH-2 interacting protein that regulates NOTCH-2 activation and angiogenesis https://hdl.handle.net/2144/23712 Boston University BOSTON UNIVERSITY SCHOOL OF MEDICINE Thesis MINAR IS A NOVEL NOTCH-2 INTERACTING PROTEIN THAT REGULATES NOTCH-2 ACTIVATION AND ANGIOGENESIS by RACHEL XI-YEEN HO B.S., University of Washington, 2015 Submitted in partial fulfillment of the requirements for the degree of Master of Science 2017 © 2017 by RACHEL XI-YEEN HO All rights reserved Approved by First Reader Nader Rahimi, Ph.D. Associate Professor, Department of Pathology & Laboratory Medicine Second Reader Dr Louis C. Gerstenfeld, Ph.D. Professor, Department of Orthopaedic Surgery ACKNOWLEDGEMENTS First, I wish like to thank Dr. Nader Rahimi, whom I look up to as a nurturing mentor and a brilliant scientist. His encouragements and philosophical approach to learning have left a great impression on me and inspired my further development as a scientist. Thank you for giving me the opportunity to study under your guidance and for helping me realize my potential. I would also like to thank all the members from the Rahimi lab, Zou, Rosana, Marwa, Rawan, Tori, Nels, and Esma, whose friendships contributed tremendously to creating a wonderful working environment. Additionally, I thank Dr. Gerstenfeld for his input on my thesis. Finally, I thank my mother and father for their unwavering love and support, and my sister for the late night conversations. This work was supported in part by grants from the NIH (R21CA191970 and R21CA193958 to N.R.) iv MINAR IS A NOVEL NOTCH-2 INTERACTING PROTEIN THAT REGULATES NOTCH-2 ACTIVATION AND ANGIOGENESIS RACHEL XI-YEEN HO ABSTRACT Angiogenesis, the formation of new vessels, is a highly regulated and complex cellular process, which plays a crucial role in physiological processes such as embryological development and wound healing.
    [Show full text]
  • C-Type Lectin CD209L/L-SIGN and CD209/DC-SIGN: Cell Adhesion Molecules Turned to Pathogen Recognition Receptors
    biology Review C-type Lectin CD209L/L-SIGN and CD209/DC-SIGN: Cell Adhesion Molecules Turned to Pathogen Recognition Receptors Nader Rahimi Department of Pathology, School of Medicine, Boston University Medical Campus, Boston, MA 02118, USA; [email protected] Simple Summary: COVID-19 pandemic continues to pose a serious threat to global public health with overwhelming worldwide socio-economic disruption. SARS-CoV-2, the viral agent of COVID- 19, uses its surface glycoprotein Spike (S) for host cell attachment and entry. The emerging picture of pathogenesis of SARS-CoV-2 demonstrates that S protein, in addition, to ACE2, interacts with the car- bohydrate recognition domain (CRD) of C-type lectin receptors, CD209L and CD209. Recognition of CD209L and CD209 which are widely expressed in SARS-CoV-2 target organs can facilitate entry and transmission leading to dysregulation of the host immune response and other major organs including, cardiovascular system. Establishing a comprehensive map of the SARS-CoV-2 interaction with CD209 family proteins, and their roles in transmission and pathogenesis can provide new insights into host-pathogen interaction with implications in therapies and vaccine development. Abstract: C-type lectin CD209/DC-SIGN and CD209L/L-SIGN proteins are distinct cell adhesion and pathogen recognition receptors that mediate cellular interactions and recognize a wide range of pathogens, including viruses such as SARS, SARS-CoV-2, bacteria, fungi and parasites. Pathogens exploit CD209 family proteins to promote infection and evade the immune recognition system. CD209L and CD209 are widely expressed in SARS-CoV-2 target organs and can contribute to in- fection and pathogenesis.
    [Show full text]
  • Cell and Molecular Biology Program, You Will Be Expected to Rotate Through Three to Four Laboratories During the Year
    Cell and Molecular Biology Program Student Handbook 2010-2011 http://www.bumc.bu.edu/cmbio www.bumc.bu.edu/gms/student-life/gmsso Contents Welcome 3 Organizational Structure 4 Academic Advisors and Student Mentors 5 Information for New Students 6 CMB Monthly Meetings 7 Rotations 8 Dates 8 Presentations 9 Rotation Evaluation Form 10 List of students in program 11 Graduates and where they are now 13 Academic Requirements and Policies 14 Research Training 18 Qualifying examinations 18 Dissertation Advisory Committee 18 Dissertation Defense 21 Pre-thesis committee evaluation forms 22 Mini Courses – Past and Present 23 Faculty that participate in CMB 25 2 Welcome to the Graduate Program The program accepted its first group of students 13 years ago and today 48 students have received their PhDs and entered industry and academia. Ten students in the program have won the coveted Henry I. Russek Achievement award and an even greater number have been awarded second place and honorable mention awards. This is a real honor! We have had 3 students win the Science Competition at the Charles River Campus. In addition, we have had one student participate in the competitive NSF program (K-12) where students teach in local school systems and take courses on learning how to teach. Many have attended local, national and international meetings and given presentations. Most importantly a large number have a great start on their CV with publications. As a member of the program you will be required to take a core set of courses in your first year. At the same time you will be doing research in a number of laboratory rotations in departments that belong to the Program.
    [Show full text]
  • The Ubiquitin-Proteasome System Meets Angiogenesis
    Published OnlineFirst February 21, 2012; DOI: 10.1158/1535-7163.MCT-11-0555 Molecular Cancer Review Therapeutics The Ubiquitin-Proteasome System Meets Angiogenesis Nader Rahimi Abstract A strict physiological balance between endogenous proangiogenic and antiangiogenic factors controls endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic angiogenesis, a shift occurs in the balance of regulators, favoring endothelial growth. Much of the control of angiogenic events is instigated through hypoxia-induced VEGF expression. The ubiquitin-proteasome system (UPS) plays a central role in fine-tuning the functions of core proangiogenic proteins, including VEGF, VEGFR- 2, angiogenic signaling proteins (e.g., the PLCg1 and PI3 kinase/AKT pathways), and other non-VEGF angiogenic pathways. The emerging mechanisms by which ubiquitin modification of angiogenic proteins control angiogenesis involve both proteolytic and nonproteolytic functions. Here, I review recent advances that link the UPS to regulation of angiogenesis and highlight the potential therapeutic value of the UPS in angiogenesis-associated diseases. Mol Cancer Ther; 11(3); 538–48. Ó2012 AACR. Introduction tumor mass reaches a macroscopic size, it is becoming Angiogenesis, the growth of new blood vessels from increasingly apparent that angiogenesis is instigated in preexisting vessels, is an important physiological process the early stage of tumor development (5, 7), further sup- in the body that is required for normal wound-healing and porting angiogenesis as a vital component of tumor female reproduction. Pathologic angiogenesis (excessive growth and metastasis. or insufficient) is now recognized as a common denom- VEGF-A also plays a central role in the development of inator underlying a number of deadly and debilitating choroidal neovascularization, and indeed is responsible human diseases, including cancer, age-related macular for both neovascularization and vascular leakage in wet degeneration (AMD), diabetic retinopathy, and cardio- AMD (8).
    [Show full text]
  • Identification of Ligand-Induced Proteolytic Cleavage and Ectodomain Shedding of VEGFR-1/FLT1 in Leukemic Cancer Cells Nader Rahimi,1 Todd E
    Published OnlineFirst March 10, 2009; DOI: 10.1158/0008-5472.CAN-08-2905 Published Online First on March 10, 2009 as 10.1158/0008-5472.CAN-08-2905 Research Article Identification of Ligand-Induced Proteolytic Cleavage and Ectodomain Shedding of VEGFR-1/FLT1 in Leukemic Cancer Cells Nader Rahimi,1 Todd E. Golde,2 and Rosana D. Meyer1 1Departments of Pathology and Ophthalmology, Boston University Medical School, Boston, Massachusetts and 2Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, Florida Abstract endothelial cells as it is poorly tyrosine phosphorylated (4–7) and Vascular endothelial growth factor receptor-1/fms-related lacks the capacity to induce gene expression (8) or to stimulate tyrosine kinase 1 (VEGFR-1/FLT1)is expressed as a mem- cellular responses such as proliferation and migration in cells with brane-bound receptor tyrosine kinase and as an alternatively endothelial origin (6, 7, 9, 10). spliced soluble protein (sVEGFR-1)containing the 1-6 IgG-like Interestingly, the expression and activation of VEGFR-1 in domain of its ectodomain. sVEGFR-1 is known as a naturally nonendothelial cells, in particular cancer cells, are linked to cell occurring inhibitor of angiogenesis and as a surrogate marker proliferation (11, 12), and a recent study indicates that prevent- for cancer progression; it is also linked to pregnancy-induced ing VEGFR-1 signaling by a VEGFR-1–specific ligand, PlGF-blocking hypertension called preeclampsia and to avascularity of nor- antibody, inhibits tumor growth in mice (13), suggesting a functional mal cornea. It remains an open question whether alternative role for VEGFR-1 signaling in tumor growth.
    [Show full text]
  • June 4, 2015 • St. Louis, Missouri
    63RD ASMS CONFERENCE ON MASS SPECTROMETRY AND ALLIED TOPICS May 31 - June 4, 2015 • St. LouiS, Missouri Welcome to the 63rd ASMS Conference on Mass Spectrometry and Allied Topics. Conference program activities and exhibit booths are in America’s Center. Corporate Member hospitality suites are located in the Renaissance Grand Hotel. SPONSORS ASMS gratefully acknowledges the support TABLE OF CONTENTS of these companies. GENERAL INFORMATION ....................................... 2 HOTELS .............................................................. 5 ASMS BOARD OF DIRECTORS ............................ 6 INTEREST GROUPS AND COMMITTEES .................. 7 AWARDS.............................................................. 8 Mobile App and WiFi CONVENTION CENTER FLOOR PLANS ................ 11 CORPORATE HOSPITALITY SUITES ..................... 13 ASMS CORPORATE MEMBERS ......................... 14 Opening Reception PROGRAM ACKNOWLEDGEMENTS ....................... 19 PROGRAM OVERVIEW ........................................ 20 WORKSHOPS ..................................................... 25 Closing Event Titles in the following sections are provided by authors. The complete abstracts are available online: www.asms.org Closing Event The PDF document of proceedings submissions for orals and posters may be viewed online one day after presentation at the conference. CONFERENCE SPONSOR SUNDAY ............................................................ 32 MONDAY ORAL SESSIONS ................................. 32 CONTRIBUTORS TUESDAY ORAL SESSIONS
    [Show full text]