VEGF Signaling: Methods and Protocols

226 Pages·2015·6.229 MB·English

by  Lorna Fiedler (eds.)

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 endothe-
lial 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 particu-
larly 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 meth-
ods 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 iso-
forms 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 sys-
tem, 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 inhib-
itors 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 car-
diovascular 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 there-
fore 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