SPRINGER LAB MANUALS

Springer-Verlag Berlin Heidelberg GmbH

Hellmut Augustin (Ed.)

Methods in Endothelial Cell Biology

With 68 Figures, 8 in Color

I i Springer

Prof.Dr. H E L L M U T G. A U G U S T I N

Department ofVascular Biology & Angiogenesis Research Tumor Biology Center at the University of Freiburg Breisacher Str. 117 79106 Freiburg Germany

e-mail: augustin@angiogenese.de

ISBN 978-3-540-21397-0 ISBN 978-3-642-18725-4 (eBook)

DOI 10.1007/978-3-642-18725-4

Library ofCongress Control Number: 2004103462

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Foreword

The field of vascular biology and its major branch, angiogenesis research,have expanded steadily since the early 1970s. Two seminal events set thesefields in motion – the publication in 1971 of the hypothesis that tumors areangiogenesis-dependent and the first isolation and culture of endothelialcells 2 years later. After decades of studies by investigators in laboratoriesthroughout the world, both fields are now solidly established. Furthermore,principles of vascular biology and angiogenesis research have been validatedin the clinic. New drugs are preventing or delaying atherosclerosis and pro-viding a novel modality for treating cancer. As a result of rapid progress inboth fields,there has been an urgent need for a book that accurately describesboth the standard methods and the new techniques being developed.It will bea valuable resource for any scientist working with endothelial cells.

Hellmut Augustin has now edited such a book. He has recruited authorswho are themselves experts and pioneers in the many aspects of vascularbiology and angiogenesis.For scientists coming into these research areas, thebook offers entrée into the breadth of subjects that can be mastered, and itwill also help those trying to adapt endothelial cell methods to suit their ownresearch. Topics include among others, isolation and analysis of circulatingendothelial precursor cells, stem cell-derived endothelial cells and lymphaticendothelial cells, plus methodologies that employ RNA-interference, confo-cal microscopy, and immortalization of endothelial cells. So, the book is avaluable contribution to these areas of research.

Methods in Endothelial Biology is also timely for another reason,the grad-ual realization by biomedical scientists and clinicians that disruptions of nor-mal endothelial cell biology underlie many diseases across the whole spec-trum of medical specialties. The United States National Institutes of Healthrecently put their imprimatur on this new paradigm by inaugurating a boldnew effort, a Trans-Institute Angiogenesis Research Program. Having a bookthat clearly and concisely embodies the experience of scientists doing vascu-lar biology and angiogenesis research is an important step into the future.

Boston, Massachusetts Judah FolkmanJune 2004 Children’s Hospital and

Harvard Medical School

Preface

The vascular endothelium lines all blood and lymphatic vessels. The morethan 1012 endothelial cells in the human body cover a surface area of morethan 1000 m2. As such, the vascular endothelium can be considered a sys-temically disseminated organ system that is directly or indirectly involvedin most of the important human diseases. Primary vascular dysfunctionscause atherosclerotic changes in the vessel wall and coagulation disorders.Inflammatory responses are controlled by chemokines and adhesion mol-ecules that act on the vessel wall or are expressed by endothelial cells,respectively. Angiogenesis is a prerequisite for the malignant growth oftumors and is also involved in numerous other physiological and patho-logical processes such as reproductive function as well as (among others)skin pathologies (e.g., psoriasis) and eye disorders (e.g., retinopathies).Lastly, vascular complications are associated with a number of chronic dis-eases, including diabetes and arthritis. Collectively, the functional involve-ment of the vessel wall in some of the most devastating human diseasesopens not only an exciting field of basic biomedical research, but alsooffers an attractive window of opportunity for the development of noveltherapeutic avenues for many diseases.

The field of vascular biology has grown rapidly within the last 20 years. Irecall my first Gordon Conference in the Summer of 1990 when I was a grad-uate student at Cornell University. I was sent to a remote place in New Hamp-shire to learn about endothelial cells. We were studying the mechanisms ofsite-specific metastasis. Significant progress had been made in the under-standing of the mechanisms of leukocyte homing and it was hypothesizedthat tumor cells similarly home to certain organs by using surface moleculesof endothelial cells as an adhesive anchor. The mechanisms of site-specificmetastasis are still largely elusive, but a lot has been learnt since then aboutthe heterogeneity and plasticity of endothelial cells in different vascularbeds. Following the (still ongoing) molecular characterization of distinct sit-uation-specific endothelial cell phenotypes such as the inflammatory or theangiogenic phenotype of endothelial cells, the molecular identification ofthe constitutive organ-specific and calibre-specific vascular body map isslowly taking shape. Advanced genomic and proteomic as well as antibodyand phage-display approaches are revolutionizing our molecular under-

standing of constitutive organotypic and calibre-specific endothelial cell dif-ferentiation. Ultimately, these experimental approaches may lead to thedevelopment of novel therapeutic strategies for the organ or site-specificdelivery of biological or pharmacological therapeutic agents. Similarly, thetherapeutic targeting of endothelial cells in distinct vascular beds, such asthe vasculature in fat tissues, is gradually taking shape.

Methods in Endothelial Cell Biology is intended to serve as a practicalhands-on guide for the continuously growing vascular biology communityin general and the endothelial cell biology community in particular. It aimsto serve as a reliable reference to some of the most important in vitro and invivo endothelial cell biology techniques that have stood the test of time orwhich have more recently evolved with great potential to further advance thediscipline. The manual is supposed to similarly provide the expert as well asthe novice in the field with a toolbox of reliable protocols to isolate and cul-ture endothelial cells, to detect endothelial cells, and to functionally manip-ulate endothelial cells in isolated cellular systems as well as in the wholebody.

When Springer first asked me to edit this manual, I was sceptical for tworeasons: First, I thought that techniques are too short-lived these days so thata manual will hardly have a reasonable half-life time. Secondly, I was con-cerned that the worldwide web with its numerous cell and molecular biolog-ical protocol collections would comprehensively cover the subject. It turnedout that I was wrong on both accounts: There is a considerable degree of tech-nical heterogeneity within the discipline. For example, more than a 1000 ves-sel density counting studies have been published in the last 15 years. Meta-analytical comparison of these studies has turned out to be a very complexchallenge as a consequence of the enormous variability of experimentalapproaches with very little standardization. Likewise, when carefully screen-ing the web with a number of different search engines for endothelial cellbiology techniques, it was surprising to see how little reliable information isactually available and that the field is far from being adequately covered.Apparently,vascular biology is still a heterogeneous community that is influ-enced by many individual organ disciplines. Yet, the increasing recognitionof the vessel wall as a systemically distributed organ system may graduallylead to a greater recognition of the need for a strong and coherent discipline.It would be a very satisfying accomplishment if Methods in Endothelial CellBiology could serve the endothelial cell biology community to contribute toa greater degree of technical coherence in the discipline.

I would like to thank the many people who supported the writing andediting of this manual. I would like to acknowledge the many colleagues,including all of the contributors of individual chapters, for their suggestionsfor topics to be covered and for their comments to the chapters. Likewise, Iwould like to thank all members of the Vascular Biology & Angiogenesis Lab-

VIII Preface

oratory at the Tumor Biology Center Freiburg, most notably Dr. UlrikeFiedler, Dr. Thomas Korff, Dr. Jens Kroll, Dr. Cynthia Obodozie, Dr. YvonneReiss, and Dr. Holger Weber for their contributions to this manual, whichwere not just restricted to the proofreading of individual chapters, but alsoto spending considerable time for practically testing many of the protocolsfor reproducibility, robustness, and reliability. Specific thanks go to ManuelaFellmann whose assistance in the editing of the manual was vital for the pro-ject. Lastly, I would like to thank Dr. Jutta Lindenborn and Friedmut Krönerfrom Springer for their patience and endurance in the production of themanual.

Freiburg, Germany Hellmut AugustinJune 2004 Tumor Biology Center Freiburg

Preface IX

Contents

Part I: Isolation and Culture of Endothelial Cells

Chapter 1 Isolation, Purification, and Culture of Human Micro- and Macrovascular Endothelial Cells . . . . . . . 3Nancy Laurens and Victor W.M. van Hinsbergh

Chapter 2 Isolation of Mouse Cardiac Microvascular Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . 15Ralf P. Brandes, Stefani Beer,

and Alexandra Aicher

Chapter 3 Isolation, Culture, and Differentiation of CirculatingEndothelial Precursor Cells . . . . . . . . . . . . . . . . 23Carmen Urbich and Stefanie Dimmeler

Chapter 4 Embryonic Stem Cell-Derived Endothelial Cells . . . . . 33Jun Yamashita and Shin-Ichi Nishikawa

Chapter 5 Detection, Isolation, and Culture Of Lymphatic Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . 47Taija Mäkinen and Kari Alitalo

Chapter 6 Three-Dimensional Spheroid Culture of Endothelial Cells . . . . . . . . . . . . . . . . . . . . . 55Thomas Korff

Chapter 7 Immortalization of Endothelial Cells . . . . . . . . . . . 63Yvonne Reiss and Friedemann Kiefer

Chapter 8 Viral Gene Transfer Into Endothelial Cells . . . . . . . . 73Christoph Volpers and Stefan Kochanek

Part II: Functional Assays in Vitro

Chapter 9 Leukocyte Adhesion to Endothelium in Vitro Under Shear Stress . . . . . . . . . . . . . . . . . . . . . 85Olaf Zille, Markus Hammel, and Rupert Hallmann

Chapter 10 Application of Biomechanical Forces on Endothelial Cells . . . . . . . . . . . . . . . . . . . . 93Andreas Schubert and Henning Morawietz

Chapter 11 Endothelial Cell Permeability Assays in Culture . . . . . 103Maria Grazia Lampugnani and Elisabetta Dejana

Chapter 12 Three-Dimensional in Vitro Angiogenesis Assays . . . . 115Thomas Korff

Chapter 13 Rat Aortic Ring Assay of Angiogenesis . . . . . . . . . . 125Roberto F. Nicosia and Wen-Hui Zhu

Chapter 14 Endothelial Cell Chemotaxis and Chemokinesis Assays . 145Hellmut G. Augustin

Chapter 15 FACS Analysis of Endothelial Cells . . . . . . . . . . . . 157Yvonne Reiss and Britta Engelhardt

Chapter 16 Application of the RNA Interference (RNAi) Technology to Angiogenesis Research . . . . . . . . . . 167Sung-Suk chae, Ji-Hye Paik,

Jonathan Shubert-Coleman,

Henry Furneaux, and Timothy Hla

Part III: Functional Assays in Vivo

Chapter 17 Murine Bone Marrow Transplantation Models that Enable the Study of EPC Recruitment . . . . . . . . 179Toshinori Murayama, Oren M. Tepper,

and Takayuki Asahara

Chapter 18 Balloon Denudation of Blood Vessels . . . . . . . . . . . 187Volkhard Lindner

XII Contents

Chapter 19 Peripheral Hind Limb Ischemia Models . . . . . . . . . 197Stephan Schirmer, Imo Hoefer, and Ivo Buschmann

Chapter 20 In Vivo Matrigel Angiogenesis Assays . . . . . . . . . . . 207Tony Passaniti and Michele I.Vitolo

Chapter 21 Analysis of Vascular Permeability in vivo . . . . . . . . . 223Katja Issbrücker, Hugo H. Marti,

and Matthias Clauss

Chapter 22 Validating the Manipulation of Endothelial Cell signaling in Vivo . . . . . . . . . . . . . . . . . . . . . . 233John S. Rudge and Gavin Thurston

Chapter 23 Chick Chorioallantoic Membrane Assay:Growth Factor and Tumor-induced Angiogenesis and Lymphangiogenesis . . . . . . . . . . . . . . . . . . 247Martin Hagedorn and Jörg Wilting

Chapter 24 Corneal Angiogenesis Assay . . . . . . . . . . . . . . . . 263 Lucia Morbidelli, Silvia Cantara,

and Marina Ziche

Part IV: Detection of Endothelial Cells

Chapter 25 Basic Blood Vessel Immunohistochemistry . . . . . . . 275Cynthia Obobodozie and Jens Kroll

Chapter 26 Double-Staining Immunohistochemistry of Blood Vessels . . . . . . . . . . . . . . . . . . . . . . . 287Valentin Goede

Chapter 27 In Situ Hybridization Analysis of Vascular Endothelium . . . . . . . . . . . . . . . . . . 301Regina Heidenreich and Georg Breier

Chapter 28 High-Resolution in Situ Confocal Analysis of Endothelial Cells . . . . . . . . . . . . . . . . . . . . . 313Holger Gerhardt and Christer Betsholtz

Contents XIII

Chapter 29 Whole Mount Analysis of the Embryonic Vasculature . . 325Jody J. Haigh and Andras Nagy

Chapter 30 Methods for Visualizing Intact Blood Vessels . . . . . . . 337Gavin Thurston and Danielle Jean-Guillaume

Chapter 31 Intravital Multi-Fluorescence Microscopy . . . . . . . . 347Mohammad Farhadi, Karsten Schwinn,

and Peter Vajkoczy

Chapter 32 Corrosion Cast Analysis of Blood Vessels . . . . . . . . . 357Valentin Djonov and Peter H. Burri

Appendix

Suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

Endothelial Cell Biology on the Internet . . . . . . . . . . . . . . . . 377

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

XIV Contents

Contributors

Aicher, Dr. Alexandra Medizinische Klinik IV,Klinikum der J.W. Goethe-UniversitätTheodor-Stern Kai 7,D-60596 Frankfurt am Main, GermanyPhone: +49-69-6301-7357,Fax: +49-69-6301-7113 E-mail: alexandra.aicher@em.uni-frankfurt.de

Alitalo, Prof. Dr. KariMolecular/Cancer Biology LaboratoryBiomedicum Helsinki and HaartmanInstitute, University of HelsinkiP.O.B. 63, SF-00014 Helsinki, FinlandPhone: +358-9-1912-5512,Fax: +358-9-1912-5510E-mail: Kari.Alitalo@Helsinki.FI

Asahara, Prof. Dr. TakayukiInstitute of Biomedical Research and Innovation / RIKEN Center for Developmental Biology2-2 Minatojima-Minamimachi, Chuo-ku,Kobe, 650-0047, JapanPhone: +81-78-304-5772,Fax: +81-78-304-5263E-mail: Asa777@aol.com

Augustin, Prof. Dr. Hellmut G.Department of Vascular Biology & Angiogenesis Research,Tumor Biology CenterBreisacher Str. 117,D-79106 Freiburg, GermanyPhone: +49-761-206-1500,Fax: +49-761-206-1505E-mail: augustin@angiogenese.de

Beer, Dr. StefaniInstitut für Kardiovaskuläre Physiologie,Universität FrankfurtTheodor-Stern Kai 7,D-60596 Frankfurt am Main, GermanyPhone: +49-69-6301-6995,Fax: +49-69-6301-7668

Betsholtz, Prof. Dr. Christer Institute of Medical Biochemistry,The Sahlgrenska Academy at GöteborgUniversity, P.O. Box 440S-405 30 Gothenburg, SwedenPhone: +46-31-773-3496,Fax: +46-31-416-108E-mail: christer.betsholtz@medkem.gu.se

Brandes, Dr. Ralf P.Institut für Kardiovaskuläre Physiologie,Universität FrankfurtTheodor-Stern Kai 7,D-60596 Frankfurt am Main, GermanyPhone: +49-69-6301-6995,Fax: +49-69-6301-7668E-mail: r.brandes@em.uni-frankfurt.de

Breier, Prof. Dr. GeorgUniversitätsklinikum Carl Gustav Carus,Institut für Pathologie, TU DresdenFetscherstr. 74, D-01307 Dresden, GermanyPhone: +49-0351-458-5278,Fax: +49-0351-458-4328E-mail: georg.breier@uniklinikum-dresden.de

Burri, Prof. Dr. Peter H.Institute of Anatomy, University of Bern,Bühlstrasse 26, CH-3012, SwitzerlandPhone: +41-31-631-4641,Fax: +41-31-631-3410E-mail: burri@ana.unibe.ch

Buschmann, Dr. IvoResearch Group for Exp. and Clin. Arteriogenesis, Dept. of Cardiology,University of FreiburgHugstetter Str. 55,D-79106 Freiburg, Germany,Phone: +49-761-270-3441,Fax:+49-761-270-3596 Email: buschmann@med1.ukl.uni-freiburg.de

Cantara, Dr. Silvia Dept. Molecular Biology,University of SienaVia Aldo Moro 2, 53100 Siena, ItalyPhone: +39-0577-234-439,Fax: +39-0577-234-343

Chae, Dr. Sung-SukCenter for Vascular Biology,University of Connecticut Health CenterFarmington, CT 06030-3501, USA Phone: +1-860-679-4128,Fax: +1-860-679-1201

Clauss, Prof. Dr. MatthiasIndiana Center for Vascular Biology &Medicine, Indiana School of Medicine975 W. Walnut Street IB 433, Indianapolis,IN 46202, USAPhone: +1-317-278-2837,Fax: +1-317-278-0089E-mail: mclauss@iupui.edu

Dejana, Prof. Dr. ElisabettaLaboratory of Vascular Biology, IFOM, FircInstitute for Molecular Oncology Via Adamello 16, I-20139 Milano, Italy Phone: +39-02574303-601,Fax: +39-02574303-244 E-mail: dejana@ifom-firc.it

Dimmeler, Prof. Dr. Stefanie Molecular Cardiology,University of FrankfurtTheodor-Stern-Kai 7,D-60596 Frankfurt/Main, GermanyPhone: +49-69-6301-7440,Fax: +49-69-6301-7113E-mail: dimmeler@em.uni-frankfurt.de

Djonov, Dr.Valentin Institute of Anatomy, University of Bern,Bühlstrasse 26, CH-3012, SwitzerlandPhone: +41-31-631-4641,Fax: +41-31-631-3410E-mail: djonov@ana.unibe.ch

Engelhardt, Prof. Dr. BrittaTheodor-Kocher Institute,University of BernFreiestr. 1, CH-3012 Bern, SwitzerlandPhone: +41-31-631-4141,Fax: +41-31-631-3799 E-mail: bengel@tki.unibe.ch

Farhadi, Dr. Mohammad Department of Neurosurgery, KlinikumMannheim, University of HeidelbergTheodor-Kutzer-Ufer 1–3,D-68167 Mannheim, GermanyPhone: +49-621-383-2360,Fax: +49-621-383-2004

Furneaux, Dr. HenryCenter for Vascular Biology,University of Connecticut Health CenterFarmington, CT 06030-3501, USA Phone: +1-860-679-4128,Fax: +1-860-679-1201

Gerhardt, Dr. Holger Institute of Medical Biochemistry,The Sahlgrenska Academy at GöteborgUniversity, P.O. Box 440S-405 30 Gothenburg, SwedenPhone: +46-31-773-3463,Fax: +46-31-416-108E-mail: holger.gerhardt@medkem.gu.se

Goede, Dr.Valentin Department of Hematology and Oncology,University Hospital CologneJoseph-Stelzmann Str. 950931 Cologne, GermanyPhone: +49-221-478-5044Fax: +49-221-478-86793E-mail: valentin.goede@uk-koeln.de

XVI Contributors

Hagedorn, Dr. MartinINSERM E0113 “Molecular mechanisms ofangiogenesis”, University of Bordeaux IAvenue des Facultes, 33405 Talence, FranceE-mail: m.hagedorn@croissance.u-bordeaux.fr

Haigh, Dr. Jody J.Department of Molecular BiomedicalResearchCardiovascular Biology UnitGhent University“Fiers-Schell-Van Montagu” buildingTechnologiepark 927B-9052 Ghent, BelgiumPhone: +32-9-33 13 730Fax: +32-9-33 13 609E-mail: Jody.Haig@dmbr.UGent.be

Hallmann, Prof. Dr. RupertDepartment of Experimental Pathology,Lund University JubileumsinstituteSoelvegatan 25, S-22362 Lund, SwedenPhone: +46-46-173419, Fax: +46-46-158202E-mail: rupert.hallmann@pat.lu.se

Hammel, Dr. Markus Department of Experimental Pathology,Lund University JubileumsinstituteSoelvegatan 25, S-22362 Lund, SwedenPhone: +46-46-173-419,Fax: +46-46-158-202E-mail: Markus.Hammel@pat.lu.se

Heidenreich, Dr. ReginaUniversitäts-Hautklinik,Eberhard-Karlos-UniversitätLiebermeisterstr. 25, D- 72076 Tübingen,GermanyPhone: +49-7071-298-4585E-mail: regina.heidenreich@med.uni-tuebingen.de

Hinsbergh, Prof. Dr.Victor W.M. vanLaboratory of Physiology,Institute of Cardiovascular Research,VU University Medical CenterVan der Boechorststraat 7,NL-1081 BT Amsterdam, The Netherlands Phone: +31-71-518-1818,Fax: +31-71-518-1904E-mail: hinsbergh@physiol.med.vu.nl

Hla, Prof. Dr. Timothy Center for Vascular Biology, University ofConnecticut Health CenterFarmington, CT 06030-3501, USA Phone: +1-860-679-4128,Fax: +1-860-679-1201E-mail: hla@nso2.uchc.edu

Hoefer, Dr. ImoPerfusion Technologies GmbHStefan-Meyer-Strasse 8, 79104 Freiburg,Germany,Phone: +49-761-203-5498 ,Fax: +49-761-203-5499 Email : mail@perfusion-technologies.com

Issbrücker, Dr. KatjaInstitute of Physiology and PathophysiologyUniversity of HeidelbergIm Neuenheimer Feld 32669120 Heidelberg, GermanyPhone: +49-6221-54-4052Fax: +49-6221-54-4561E-mail: k.issbrueckeer@pio1.uni-heidelberg.de

Jean-Guillaume, Dr. DanielleRegeneron Pharmaceuticals Inc.777 Old Saw Mill River Rd., Tarrytown,NY 10591, USAPhone: +1-914-997-9881,Fax: +1-914-347-5045

Kiefer, Dr. Friedemann Department of Vascular Cell Biology,Max-Planck-Institute for Vascular BiologyVon-Esmarch-Straße 56,D-48149 Münster GermanyPhone: +49-251-835-2221,Fax: +49-251-835-8616 Email: fkiefer@gwdg.de

Kochanek, Prof. Dr. StefanSektion GentherapieUniversität UlmHelmholtz Str. 8/1D-89081 Ulm, GermanyPhone: +49-731-500-33647Fax: +49-731-500-33664E-mail: stefan.kochanek@medizin.uni-ulm.de

Contributors XVII

Korff, Prof. Dr. ThomasAbt. Herz- und Kreislaufphysiologie,Universität GöttingenHumboldtallee 23,D-37073 Göttingen, GermanyPhone: +49-551-39-5891,Fax: +49-551-39-5883Email: korff@veg-physiol.med.uni-goettingen.de

Kroll, Dr. JensDepartment of Vascular Biology & Angio-genesis Research, Tumor Biology CenterBreisacher Str. 117,D-79106 Freiburg, GermanyPhone: +49-761-206-1501,Fax: +49-761-206-1505E-mail: kroll@tumorbio.uni-freiburg.de

Lampugnani, Dr. Maria Grazia Laboratory of Vascular Biology, IFOM, FircInstitute for Molecular Oncology Via Adamello 16, I-20139 Milano, Italy Phone: +39-02574303-601,Fax: +39-02574303-244 email: lampugnani@ifom-firc.it

Laurens, Dr. NancyDepartment of Vascular and ConnectiveTissue Research, Gaubius Laboratory,TNO-PGZernikedreef 9, NL-2333 CK Leiden,The Netherlands

Lindner, Prof. Dr.Volkhard Center for Molecular Medicine, MaineMedical Center Research Institute81 Research Drive, Scarborough,ME 04074, USAPhone: +1-207-885-8143,Fax: +1-207-885-8179E-mail: lindnv@mmc.org

Mäkinen, Dr. TaijaDepartment of Molecular Neurobiology,Max-Planck-Institute of NeurobiologyAm Klopferspitz 18A,D-82152 Martinsried, GermanyPhone: +49-89-8578-3187,Fax: +49-89-8578-3152E-mail: makinen@neuro.mpg.de

Marti, Prof. Dr. Hugo H.Institut für Physiologie und Patho-physiologie, Universität HeidelbergIm Neuenheimer Feld 326,D-69 120 Heidelberg, GermanyPhone: +49-6221-544-035,Fax: +49-6221-544-038E-mail: hugo.marti@pio1.uni-heidelberg.de

Morawietz, Prof. Dr. HenningDepartment of Vascular Endothelium and Microcirculation,University of Technology DresdenFetscherstrasse 74,D-01307 Dresden, GermanyPhone +49-345-557-1454,Fax +49-345-557-1404E-mail: henning.morawietz@mailbox.tu-dresden.de

Morbidelli, Dr. Lucia Dept. Molecular Biology,University of SienaVia Aldo Moro 2, 53100 Siena, ItalyPhone: +39-0577-234-439,Fax: +39-0577-234-343E-mail: morbidelli@unisi.it

Murayama, Dr. Toshinori Translational Research Center,Kyoto University Hospital54 Shogoin-Kawaharacho, Kyoto 606-8507,Japan

Nagy, Prof. Dr. Andras Samuel Lunenfeld Research Institute,Mount Sinai Hospital600 University Avenue, Toronto, Ontario,CanadaPhone: +1-416-586-8455,Fax: +1-416-586-8588E-mail: nagy@mshri.on.ca

Nicosia, Prof. Dr. Roberto F.Division of Pathology and LaboratoryMedicine,Veterans Affairs Puget SoundHealth Care SystemSeattle, WA, USAPhone: +1-206-764-2284,Fax: +1-206-764-2001E-mail: Roberto.Nicosia@med.va.gov

XVIII Contributors

Nishikawa, Prof. Dr. Shin-IchiStem Cell Biology Laboratory,Center for Developmental BiologyRiken 2-2-3, Minatojima-minamimachi,Chuou-ku, Kobe 650-0047, JapanPhone: +81-78-306-1893,Fax: +81-78-306-1895E-mail: nishikawa@cdb.riken.go.jp

Obodozie, Dr. CynthiaDepartment of Vascular Biology & Angio-genesis Research, Tumor Biology CenterBreisacher Str. 117,D-79106 Freiburg, GermanyPhone: +49-761-206-1501,Fax: +49-761-206-1505E-mail: obodozie@tumorbio.uni-freiburg.de

Paik, Dr. Ji-HyeCenter for Vascular Biology,University of Connecticut Health CenterFarmington, CT 06030-3501, USA Phone: +1-860-679-4128,Fax: +1-860-679-1201E-mail: paik@student.uchc.edu

Passaniti, Prof. Dr. ToniDepartment of Pathology, Biochemistry &Molecular Biology, University of MarylandGreenebaum Cancer Center,Bressler Research Building, Room 7-021655 W. Baltimore St., Baltimore,MD 21201, USAPhone: +1-410-328-5470,Fax: +1-410-328-6559E-mail: apass001@umaryland.edu

Reiss, Dr.Yvonne Department of Vascular Biology & Angio-genesis Research, Tumor Biology CenterBreisacher Str. 117,D-79106 Freiburg, GermanyPhone: +49-761-206-1510,Fax: +49-761-206-1505E-mail: reiss@tumorbio.uni-freiburg.de

Rudge, Dr. John S.Regeneron Pharmaceuticals Inc.777 Old Saw Mill River Rd., Tarrytown,NY 10591, USAPhone: +1-914-997-9881,Fax: +1-914-347-5045E-mail: John.Rudge@Regeneron.com

Schirmer, Dr. StephanResearch Group for Exp. and Clin. Arteriogenesis, Dept. of Cardiology,University of FreiburgHugstetter Str. 55,D-79106 Freiburg, Germany,Phone: +49-761-270-3441,Fax:+49-761-270-3596 E-mail: schirmer@med1.ukl.uni-freiburg.de

Schubert, Dr. Andreas Heart Center, University LeipzigRussenstrasse 19,D-04289 Leipzig, GermanyPhone: +49-341-865-1620,Fax: +49-341-865-1452E-mail: schua@medizin.uni-leipzig.de

Schwinn, Dr. Karsten Department of Neurosurgery,Klinikum Mannheim,University of HeidelbergTheodor-Kutzer-Ufer 1-3,D-68167 Mannheim, GermanyPhone: +49-621-383-2360,Fax: +49-621-383-2004

Shubert-Coleman, Dr. JonathanCenter for Vascular Biology,University of Connecticut Health CenterFarmington, CT 06030-3501, USA Phone: +1-860-679-4128,Fax: +1-860-679-1201

Tepper, Dr. Oren M.New York University Medical Center550 First Avenue, New York NY 10016-6497, USAE-mail: teppeo01@med.nyu.edu

Contributors XIX

Thurston, Dr. Gavin Regeneron Pharmaceuticals Inc.777 Old Saw Mill River Rd., Tarrytown,NY 10591, USAPhone: +1-914-997-9881,Fax: +1-914-347-5045E-mail: gavin.thurston@regeneron.com

Urbich, Dr. Carmen Molecular Cardiology,University of FrankfurtTheodor-Stern-Kai 7,D-60596 Frankfurt/Main, GermanyPhone: +49-69-6301-7440,Fax: +49-69-6301-7113E-mail: urbich@em.uni-frankfurt.de

Vajkoczy, Dr. Peter Department of Neurosurgery, KlinikumMannheim, University of HeidelbergTheodor-Kutzer-Ufer 1-3,D-68167 Mannheim, GermanyPhone: +49-621-383-2360,Fax: +49-621-383-2004E-mail: peter.vajkoczy@nch.ma.uni-heidelberg.de

Vitolo, Dr. Michele I.Department of Biochemistry & MolecularBiology, University of Maryland School of Medicine655 W. Baltimore St., Baltimore,MD 21201, USAE-mail: mvitolo@umaryland.edu

Volpers, Dr. Christoph CEVEC Pharmaceuticals GmbHGottfried-Hagen-Str. 6251105 Cologne, GermanyPhone: +49-221-46020800Fax: +49-221-46020801E-mail: volpers@cevec-pharmaceuticals.com

Wilting, Prof. Dr. JörgPediatrics I, Children’s Hospital,University of GöttingenRobert-Koch-Strasse 40,D-37075 Göttingen, GermanyPhone: +49-551-39-6270,Fax: +49-551-39-6231E-mail: joerg.wilting@med.uni-goettingen.de

Yamashita, Dr.YunDepartment of Molecular Genetics,Kyoto University Graduate School of Medicine53 Shogoin Kawahara-cho,Sakyo, Kyoto 606-8507, JapanPhone: +81-75-751-4163,Fax: +81-75-751-4169E-mail: juny@kuhp.kyoto-u.ac.jp

Zhu, Dr. Wen-HuiDepartment of Pathology,University of WashingtonSeattle, WA 98195-7470 , USA

Ziche, Prof. Dr. MarinaDept. Molecular Biology,University of SienaVia Aldo Moro 2, 53100 Siena, ItalyPhone: +39-0577-234-439,Fax: +39-0577-234-343E-mail: ziche@unisi.it

Zilles, Dr. OlafDepartment of Experimental Pathology,Lund University JubileumsinstituteSoelvegatan 25, S-22362 Lund, SwedenPhone: +46-46-173419,Fax: +46-46-158202E-mail: ozilles@gmx.de

XX Contributors