Table Of Content

Methods in Molecular Biology 1332 Lorna Fiedler Editor VEGF Signaling Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 VEGF Signaling Methods and Protocols Edited by Lorna Fiedler Caversham, Reading, UK Editor Lorna Fiedler Caversham Reading, UK ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2916-0 ISBN 978-1-4939-2917-7 (eBook) DOI 10.1007/978-1-4939-2917-7 Library of Congress Control Number: 2015945619 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2 015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Prefa ce Since its discovery in the 1980s as a potent endothelial cell growth and permeability inducer, there has been an explosion of research into the role of VEGF (vascular endothelial growth factor or vascular permeability factor) in physiology, pathology, and therapy. VEGF is indispensable for cardiovascular and lymphatic development, and in physiological angiogenesis in the adult (pregnancy, menstruation, and wound healing for example). However its dysregulation underlies multiple diseases including diabetic complications, rheumatoid arthritis, heart failure, hypertension, metastatic cancer, macular degeneration, renal disease, and pre-eclampsia. VEGF and its activators or effectors are therefore particularly attractive therapeutic targets; however more failures than successes have been achieved in clinical translation and it is therefore imperative to understand the cellular mechanisms and consequences involved. This volume provides a collection of protocols for studying and manipulating VEGF signaling pathways in vitro and in vivo, and in particular, aims to present a range of both fi rmly established and newly emerging technologies, including those that are amenable to aiding in drug discovery or preclinical investigations. Each part of the three begins with an introductory overview followed by relevant methods chapters; of particular importance, and a major advantage of this series in general, each chapter contains not only methodological detail rarely seen in the literature but also a sec- tion on key notes and troubleshooting advice. Part I focuses on quantifi cation of specifi c VEGF and VEGF receptor isoforms, beginning with an overview on VEGF splice variants, their function, and regulation (particularly pre-mRNA splicing). Methods for mechanically inducing VEGF expression (mimicking changes in extracellular matrix rigidity), studying promoter–transcription factor interactions, and quantifying VEGF and VEGF receptor isoforms follow. Part II relates to the study of VEGF-dependent signaling in vitro, beginning with a comprehensive overview of VEGF-mediated signal transduction in the cardiovascular system, highlighting recent discoveries in the fi eld. This part contains methods for monitoring ligand-stimulating VEGF receptor traffi cking, cell-based screening of small molecule inhibitors directed against VEGF receptors, assaying ERK5 activity (a more recently identifi ed kinase particularly associated with VEGF-dependent effects), and studying VEGF- dependent cell motility and in vitro angiogenesis. Part III details selected in vivo models. A summary of genetic mouse models is fi rst presented, focusing on methodological approaches for understanding the role of VEGFR-1/ Flt-1 in the cardiovascular system. Techniques for studying physiological angiogenesis in the developing mouse embryo, particularly amenable to genetic and pharmacological stud- ies, are also detailed. Although mouse models have been an unequivocal tool in scientifi c research, larger mammals may be more representative of certain aspects of the human cardiovascular system. Novel techniques for gene transfer into sheep and guinea pigs that have been used to study VEGF gene therapy in pregnancy and obstetric complications are therefore presented. Non-mammalian species like the zebrafi sh are often used in basic f undamental research into gene function. A novel method is presented for generating targeted mutations v vi Preface using the CRISPR/Cas system. Although focused on the zebrafi sh, this technology can easily be adapted to other model organisms, and signifi cantly, this technology has not yet been applied to the VEGF pathway and no doubt will become an indispensable approach in the future. I hope that these methods and approaches will provide a useful tool for research in the fi eld, in understanding the basic biology of VEGF signaling and in translating this research into the clinic. This would not have been possible without everyone who has contributed to this book, as well as the series editor, John Walker, who has provided a constant source of support and to whom I am particularly grateful. Finally, I would like to thank all the authors for their hard work and contributions, and I hope that researchers, both “young” and “old,” will fi nd this book useful. Reading, U K L orna Fiedler Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x PART I VEGF AND VEGF RECEPTORS 1 VEGF Splicing and the Role of VEGF Splice Variants: From Physiological-Pathological Conditions to Specific Pre-mRNA Splicing. . . . . 3 Mélanie Guyot and Gilles P agès 2 D etection and Quantification of VEGF Isoforms by ELISA. . . . . . . . . . . . . . . 2 5 Jean-Michel V ernes and Y. G loria Meng 3 Q uantitation of Circulating Neuropilin-1 in Human, Monkey, Mouse, and Rat Sera by ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 Yanmei Lu and Y . G loria M eng 4 D etection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells . . . . . . . . . . . 4 9 Gareth W. Fearnley , Stephen B. W heatcroft , and Sreenivasan P onnambalam 5 I nduction of VEGF Secretion in Cardiomyocytes by Mechanical Stretch . . . . . 67 Michelle L. M atter 6 Chromatin Immunoprecipitation Assay: Examining the Interaction of NFkB with the VEGF Promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5 Chad B . Walton and M ichelle L. Matter PART II M ODULATION AND ANALYSIS OF VEGF DEPENDENT SIGNALING 7 An Overview of VEGF-Mediated Signal Transduction. . . . . . . . . . . . . . . . . . . 9 1 Ian E vans 8 I dentification of Receptor Tyrosine Kinase Inhibitors Using Cell Surface Biotinylation and Affinity Isolation . . . . . . . . . . . . . . . . . . . . . . . 121 Antony M . Latham , Jayakanth K ankanala , C olin W. G. F ishwick , and S reenivasan P onnambalam 9 Analysis of VEGF-Mediated ERK5 Activity in Endothelial Cells . . . . . . . . . . . 1 33 Gopika N. N ithianandarajah-Jones and Michael J. Cross 10 In Vitro Angiogenesis Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Ian E vans 11 C hemotactic Migration of Endothelial Cells Towards VEGF-A . . . . . . . . . . 151 165 Caroline Pellet-Many vii viii Contents PART III IN VIVO MODELS TO STUDY VEGF SIGNALING 12 Vasculogenesis and Angiogenesis in VEGF Receptor-1 Deficient Mice. . . . . . . 1 61 Vivienne C . Ho and Guo-Hua F ong 13 T he Embryonic Mouse Hindbrain and Postnatal Retina as In Vivo Models to Study Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 77 Alessandro Fantin and C hristiana R uhrberg 14 VEGF Gene Transfer to the Utero-Placental Circulation of Pregnant Guinea Pigs to Enhance Fetal Growth . . . . . . . . . . . . . . . . . . . . . 189 Vedanta Mehta , David J . C arr , A nna Swanson , and Anna L. David 15 V EGF Gene Transfer to the Utero-Placental Circulation of Pregnant Sheep to Enhance Fetal Growth. . . . . . . . . . . . . . . . . . . . . . . . . . 197 David J . C arr , V edanta M ehta , J acqueline M. W allace , and Anna L . D avid 16 G eneration of Targeted Mutations in Zebrafish Using the CRISPR/Cas System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 05 Linlin Y in , Li-En J ao , and Wenbiao C hen Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 19 Contributors DAVID J. C ARR • Prenatal Cell and Gene Therapy Group, U CL Institute for Women’s Health, University College London , L ondon, UK ; Rowett Institute of Nutrition and Health, U niversity of Aberdeen , A berdeen, U K WENBIAO C HEN • Department of Molecular Physiology and Biophysics, V anderbilt University School of Medicine , Nashville, T N , U SA MICHAEL J. CROSS • Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, Institute of Translational Medicine , U niversity of Liverpool , Liverpool, U K ANNA L . DAVID • Institute for Women’s Health, University College London , L ondon, UK IAN EVANS • Centre for Cardiovascular Biology and Medicine, Division of Medicine, University College London , London, UK ALESSANDRO F ANTIN • UCL Institute of Ophthalmology , University College London , London, U K GARETH W . FEARNLEY • Endothelial Cell Biology Unit, School of Molecular and Cellular Biology , U niversity of Leeds , Leeds, U K COLIN W. G. FISHWICK • School of Chemistry , University of Leeds , L eeds , U K GUO-HUA FONG • Department of Cell Biology, Center for Vascular Biology , U niversity of Connecticut Health Center , Farmington, CT , U SA MÉLANIE GUYOT • Institute for Research on Cancer and Aging of Nice (IRCAN), University of Nice Sophia Antipolis , Nice Cedex, France VIVIENNE C. HO • Department of Cell Biology, Center for Vascular Biology , University of Connecticut Health Center , Farmington, C T , USA LI-EN JAO • Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis , Davis, C A , USA JAYAKANTH KANKANALA • School of Chemistry , U niversity of Leeds , L eeds, UK ANTONY M . LATHAM • Endothelial Cell Biology Unit, School of Molecular and Cellular Biology , University of Leeds , L eeds, UK YANMEI LU • Department of Biochemical & Cellular Pharmacology, Research and Early Development, G enentech Inc. , S outh San Francisco, CA , U SA MICHELLE L. M ATTER • University of Hawaii Cancer Center , H onolulu, H I , U SA ; John A. Burns School of Medicine , Honolulu, H I , USA VEDANTA MEHTA • Institute for Women’s Health, U niversity College London , L ondon, UK Y. GLORIA MENG • Department of Biochemical & Cellular Pharmacology, Research and Early Development,, G enentech Inc. , South San Francisco, C A , U SA GOPIKA N . NITHIANANDARAJAH-JONES • Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool , Liverpool, U K GILLES PAGÈS • Institute for Research on Cancer and Aging of Nice (IRCAN), University of Nice Sophia Antipolis , N ice Cedex, F rance ix