The Blood-Brain and Other Neural Barriers: Reviews and Protocols (Methods in Molecular Biology, Volume 686)

Methods

in

Molecular Biology™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK



For other titles published in this series, go to www.springer.com/series/7651

The Blood-Brain and Other Neural Barriers Reviews and Protocols

Edited by

Sukriti Nag University of Toronto, Toronto, ON, Canada

Editor Sukriti Nag, MD, Ph.D. Department of Laboratory Medicine and Pathobiology University of Toronto Toronto, ON Canada [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-937-6 e-ISBN 978-1-60761-938-3 DOI 10.1007/978-1-60761-938-3 Springer New York Dordrecht Heidelberg London © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or ­dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, ­neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover Illustration: Modified merged confocal image showing components of the neurovascular unit. Image provided by Anish Kapadia and Sukriti Nag. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface More than 100 years have elapsed since the discovery of the blood-brain barrier (BBB). Evolving technologies starting with tracer studies, and more recently with genomics and proteomics, have provided novel information about the molecular properties of cerebral endothelium and astrocytes. The concept of the neurovascular unit has provided an impetus for in  vitro studies of the interaction of brain endothelial cells with other components of the neurovascular unit such as pericytes, astrocytes, and neurons in steady states. However, such studies have to be done in animal models of neurological diseases and in humans to get a clearer understanding of the pathogenesis of BBB breakdown in nervous system diseases. Determination of the temporal course of BBB breakdown and the parallel molecular alterations remain important goals to identify therapeutic windows and pertinent therapeutic agents which will modify the disease process and prevent irreversible brain damage. There is also the need to develop imaging techniques for early diagnosis of brain diseases before irreparable tissue damage results. Although, modest advances have been made in the area of the BBB, parallel advances have not been made in the other neural barriers. These need to be studied to obtain an overall picture of the disease process. The Blood-Brain and Other Neural Barriers: Reviews and Protocols, a sequel to The Blood-Brain Barrier: Biology and Research Protocols, provides the reader with additional protocols to study the barriers. The first section consists of current reviews of the properties of some of the components of the neurovascular unit, namely the brain endothelium, pericytes, and astrocytes. In addition, current information about the blood- cerebrospinal fluid barrier, the blood-retinal and blood-nerve barriers is also provided. The second section of the book gives detailed protocols of specific techniques written by experts in the field. The protocols include applications as well as caveats of these techniques. The first part describes techniques to image the barriers in humans and experimental animals, followed by cutting-edge molecular techniques to study the BBB and novel models to study the barriers. The last part details some of the prevalent techniques for the delivery of therapeutic agents across the BBB. This is a rapidly growing and competitive field, and, in some cases, results are too preliminary for publication of detailed protocols. It is hoped that the detailed protocols given in this book will aid the research efforts of not only graduate students but also more experienced investigators and will enable more studies of the blood-cerebrospinal, blood-retinal, and blood-nerve barriers. I would like to acknowledge Prof. John M. Walker for this opportunity and for his help and all the authors who have contributed their protocols without which this book would not be possible. This book is dedicated to my parents, Mohit Kumar and Labonya Nag for their unconditional love and support and for giving me the opportunity to pursue my goals.

Sukriti Nag Toronto, ON

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Part I  Biology of the Barriers   1 Morphology and Properties of Brain Endothelial Cells . . . . . . . . . . . . . . . . . . . . . 3 Sukriti Nag   2 Morphology and Properties of Pericytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Paula Dore-Duffy and Kristen Cleary   3 Morphology and Properties of Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Sukriti Nag   4 The Blood–Cerebrospinal Fluid Barrier: Structure and Functional Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Conrad E. Johanson, Edward G. Stopa, and Paul N. McMillan   5 The Blood-Retinal Barrier: Structure and Functional Significance . . . . . . . . . . . . 133 E. Aaron Runkle and David A. Antonetti   6 The Blood-Nerve Barrier: Structure and Functional Significance . . . . . . . . . . . . . 149 Ananda Weerasuriya and Andrew P. Mizisin

Part II  Imaging the Barriers   7 Detection of Multiple Proteins in Intracerebral Vessels by Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Janet L. Manias, Anish Kapadia, and Sukriti Nag   8 Multiparametric Magnetic Resonance Imaging and Repeated Measurements of Blood-Brain Barrier Permeability to Contrast Agents . . . . . . . . . . . . . . . . . . . . Tavarekere N. Nagaraja, Robert A. Knight, James R. Ewing, Kishor Karki, Vijaya Nagesh, and Joseph D. Fenstermacher   9 Detection of Brain Pathology by Magnetic Resonance Imaging of Iron Oxide Micro-particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel C. Anthony, Nicola R. Sibson, Martina A. McAteer, Ben Davis, and Robin P. Choudhury 10 Measuring the Integrity of the Human Blood-Brain Barrier Using Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrea Kassner and Rebecca Thornhill 11 Assessing Blood–Cerebrospinal Fluid Barrier Permeability in the Rat Embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Norman R. Saunders, C. Joakim Ek, Mark D. Habgood, Pia Johansson, Shane Liddelow, and Katarzyna M. Dziegielewska

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12 Detection of Blood–Nerve Barrier Permeability by Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Carsten Wessig

Part III Molecular Techniques to Study the Blood-Brain Barrier 13 Isolation of Human Brain Endothelial Cells and Characterization of Lipid Raft-Associated Proteins by Mass Spectroscopy . . . . . . . . . . . . . . . . . . . . Romain Cayrol, Arsalan S. Haqqani, Igal Ifergan, Aurore Dodelet-Devillers, and Alexandre Prat 14 Analysis of Mouse Brain Microvascular Endothelium Using Laser Capture Microdissection Coupled with Proteomics . . . . . . . . . . . . . . . . . . . . . . . Nivetha Murugesan, Jennifer A. Macdonald, Qiaozhan Lu, Shiaw-Lin Wu, William S. Hancock, and Joel S. Pachter 15 Molecular and Functional Characterization of P-Glycoprotein In Vitro . . . . . . . . Gary N. Y. Chan and Reina Bendayan 16 Methods to Study Glycoproteins at the Blood-Brain Barrier Using Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arsalan S. Haqqani, Jennifer J. Hill, James Mullen, and Danica B. Stanimirovic

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Part IV Models to Study the Barriers 17 Novel Models for Studying the Blood-Brain and Blood-Eye Barriers in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert L. Pinsonneault, Nasima Mayer, Fahima Mayer, Nebiyu Tegegn, and Roland J. Bainton 18 Zebrafish Model of the Blood-Brain Barrier: Morphological and Permeability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brian P. Eliceiri, Ana Maria Gonzalez, and Andrew Baird 19 Methods to Assess Pericyte-Endothelial Cell Interactions in a Coculture Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gokulan Thanabalasundaram, Jehad El-Gindi, Mira Lischper, and Hans-Joachim Galla 20 Isolation and Properties of an In Vitro Human Outer Blood-Retinal Barrier Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robin D. Hamilton and Lopa Leach 21 Isolation and Properties of Endothelial Cells Forming the Blood-Nerve Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yasuteru Sano and Takashi Kanda

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Part V Delivery of Therapeutic Agents Across the Barriers 22 Treatment of Focal Brain Ischemia with Viral Vector-Mediated Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 Hua Su and Guo-Yuan Yang 23 Blood-Brain Barrier Disruption in the Treatment of Brain Tumors . . . . . . . . . . . . 447 Marie Blanchette and David Fortin

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24 Integrated Platform for Brain Imaging and Drug Delivery Across the Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Umar Iqbal, Abedelnasser Abulrob, and Danica B. Stanimirovic 25 Targeting the Choroid Plexus-CSF-Brain Nexus Using Peptides Identified by Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 Andrew Baird, Brian P. Eliceiri, Ana Maria Gonzalez, Conrad E. Johanson, Wendy Leadbeater, and Edward G. Stopa Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

Contributors Abedelnasser Abulrob  •  Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada Daniel C. Anthony  •  Departments of Pharmacology and Chemistry, University of Oxford, Oxford, UK David A. Antonetti  •  Department of Ophthalmology, Penn State College of Medicine, Hershey, PA, USA Roland J. Bainton  •  Department of Anesthesia and Perioperative Care, San Francisco General Hospital, University of California at San Francisco, San Francisco, CA, USA Andrew Baird  •  Department of Surgery, University of California San Diego, San Diego, CA, USA Reina Bendayan  •  Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada Marie Blanchette  •  Departments of Neurosurgery and Neuro-oncology, University of Sherbrooke Hospital, Sherbrooke, QC, Canada Romain Cayrol  •  Neuroimmunology Research Laboratory, CHUM-Notre-Dame Hospital, Université de Montréal, Montréal, QC, Canada Gary N.Y. Chan  •  Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada Robin P. Choudhury  •  Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Kristen Cleary  •  Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA Ben Davis  •  Departments of Pharmacology and Chemistry, University of Oxford, Oxford, UK Aurore Dodelet-Devillers  •  Neuroimmunology Research Laboratory, CHUM-Notre-Dame Hospital, Université de Montréal, Montréal, QC, Canada Paula Dore-Duffy  •  Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA Katarzyna M. Dziegielewska  •  Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia C. Joakim Ek  •  Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia Brian P. Eliceiri  •  Department of Surgery, University of California San Diego, San Diego, CA, USA Jehad El-Gindi  •  Institute of Biochemistry, Westfälische Wilhelms Universität Münster, Münster, Germany James R. Ewing  •  Department of Neurology, Henry Ford Hospital, Detroit, MI, USA

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Contributors

Joseph D. Fenstermacher  •  Department of Anesthesiology, Henry Ford Hospital, Detroit, MI, USA David Fortin  •  Departments of Neurosurgery and Neuro-oncology, University of Sherbrooke Hospital, Sherbrooke, QC, Canada Hans-Joachim Galla  •  Institute of Biochemistry, Westfälische Wilhelms Universität Münster, Münster, Germany Ana Maria Gonzalez  •  Molecular Neuroscience Group, School of Medicine, University of Birmingham, Edgbaston, Birmingham, UK Mark D. Habgood  •  Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia Robin D. Hamilton  •  School of Biomedical Sciences, University of Nottingham, Nottingham, UK William S. Hancock  •  Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA, USA Arsalan S. Haqqani  •  Proteomics Group, Institute of Biological Sciences, National Research Council, Ottawa, ON, Canada Jennifer J. Hill  •  Proteomics Group, Institute of Biological Sciences, National Research Council, Ottawa, ON, Canada Igal Ifergan  •  Neuroimmunology Research Laboratory, CHUM-Notre-Dame Hospital, Université de Montréal, Montréal, QC, Canada Umar Iqbal  •  Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada Conrad E. Johanson  •  Department of Clinical Neuroscience, Alpert Medical School at Brown University, Providence, RI, USA Pia Johansson  •  Institute for Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany Takashi Kanda  •  Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan Anish Kapadia  •  Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Kishor Karki  •  Department of Neurology, Henry Ford Hospital, Detroit, MI, USA Andrea Kassner  •  Department of Medical Imaging, University of Toronto, Toronto, ON, Canada Robert A. Knight  •  Department of Neurology, Henry Ford Hospital, Detroit, MI, USA Lopa Leach  •  School of Biomedical Sciences, University of Nottingham, Nottingham, UK Wendy Leadbeater  •  Molecular Neuroscience Group, School of Medicine, University of Birmingham, Edgbaston, Birmingham, UK Shane Liddelow  •  Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia Mira Lischper  •  Institute of Biochemistry, Westfälische Wilhelms Universität Münster, Münster, Germany Qiaozhan Lu  •  Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA, USA

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Jennifer A. Macdonald  •  Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA Janet L. Manias  •  Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Fahima Mayer  •  Department of Anesthesia and Perioperative Care, San Francisco General Hospital, University of California at San Francisco, San Francisco, CA, USA Nasima Mayer  •  Department of Anesthesia and Perioperative Care, San Francisco General Hospital, University of California at San Francisco, San Francisco, CA, USA Martina A. Mcateer  •  Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Paul N. Mcmillan  •  Department of Pathology, Alpert Medical School at Brown University, Providence, RI, USA Andrew P. Mizisin  •  Department of Pathology, School of Medicine, University of California San Diego, San Diego, CA, USA James Mullen  •  Proteomics Group, Institute of Biological Sciences, National Research Council, Ottawa, ON, Canada Nivetha Murugesan  •  Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA Sukriti Nag  •  Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Tavarekere N. Nagaraja  •  Department of Anesthesiology, Henry Ford Hospital, Detroit, MI, USA Vijaya Nagesh  •  Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA Joel S. Pachter  •  Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA Robert L. Pinsonneault  •  Department of Anesthesia and Perioperative Care, San Francisco General Hospital, University of California at San Francisco, San Francisco, CA, USA Alexandre Prat  •  Department of Neurology, Neuroimmunology Research Laboratory, CHUM-Notre-Dame Hospital, Université de Montréal, Montréal, QC, Canada E. Aaron Runkle  •  Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA, USA Yasuteru Sano  •  Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan Norman R. Saunders  •  Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia Nicola R. Sibson  •  Experimental Neuroimaging Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Danica B. Stanimirovic  •  Proteomics Group, Institute of Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada Edward G. Stopa  •  Department of Pathology, Alpert Medical School at Brown University, Providence, RI, USA

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Contributors

Hua Su  •  Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, CA, USA Nebiyu Tegegn  •  Department of Anesthesia and Perioperative Care, San Francisco General Hospital, University of California at San Francisco, San Francisco, CA, USA Gokulan Thanabalasundaram  •  Institute of Biochemistry, Westfälische Wilhelms Universität Münster, Münster, Germany Rebecca Thornhill  •  Department of Medical Imaging, University of Toronto, Toronto, ON, Canada Ananda Weerasuriya  •  Division of Basic Medical Sciences, School of Medicine, Mercer University, Macon, GA, USA Carsten Wessig  •  Department of Neurology, University of Würzburg, Würzburg, Germany Shiaw-Lin Wu  •  Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA, USA Guo-Yuan Yang  •  Med-X Research Institute, Sanghai JiaoTong University, Shanghai, China; Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California at San Francisco, San Francisco, CA, USA

Part I Biology of the Barriers

Chapter 1 Morphology and Properties of Brain Endothelial Cells Sukriti Nag Abstract The molecular advances in various aspects of brain endothelial cell function in steady states are considerable and difficult to summarize in one chapter. Therefore, this chapter focuses on endothelial permeability mechanisms in steady states and disease namely vasogenic edema. The morphology and properties of caveolae and tight junctions that are involved in endothelial permeability to macromolecules are reviewed. Endothelial transport functions are briefly reviewed. Diseases with alterations of endothelial permeability are mentioned and details are provided of the molecular alterations in caveolae and tight junctions in vasogenic edema. Other factors involved in increased endothelial permeability such as the matrix metalloproteinases are briefly discussed. Of the modulators of endothelial permeability, angioneurins such as the vascular endothelial growth factors and angiopoietins are discussed. The chapter concludes with a brief discussion on delivery of therapeutic substances across endothelium. Key words: Adherens junctions, Angioneurins, Angiopoietin 1, Angiopoietin 2, Blood-brain barrier, Brain endothelium, Carrier-mediated transport, Caveolae, Caveolin-1, Claudins, Cortical coldinjury model, Efflux transport, Junctional adhesion molecule-1, Matrix metalloproteinases, Occludin, Receptor-mediated transport, Tight junctions, Transcytosis, Vascular endothelial growth factor-A, Vascular endothelial growth factor-B, Vasogenic edema, Zonula occludens

1. Introduction The recognition of a restrictive barrier that limits the passage of substances from the blood into the brain dates back to the work of Paul Ehrlich (1), who injected coerulean-S sulfate intravenously into rodents and observed that all the body organs were stained blue except for the brain. These findings were confirmed by other investigators and the concept of a blood-brain barrier (BBB) arose. Ongoing research led to the discovery of the transport properties of endothelium and a broader definition of the BBB emerged which included anatomical, physicochemical, and

Sukriti Nag (ed.), The Blood-Brain and Other Neural Barriers: Reviews and Protocols, Methods in Molecular Biology, vol. 686, DOI 10.1007/978-1-60761-938-3_1, © Springer Science+Business Media, LLC 2011

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biochemical mechanisms at the level of the endothelium, which influence the exchange of materials between blood and brain and cerebrospinal fluid. There is growing recognition that integrated brain function and dysfunction arise from the complex interactions between a network of multiple cell types, such as neurons, glial cells including astrocytes, oligodendrocytes, microglia, and components of the brain vasculature including endothelial cells, smooth muscle cells, and pericytes. The interaction of these cells in steady states and their coordinated response to injury led to the concept that these cells constitute a functional unit, termed the neurovascular unit (2, 3) (Fig. 1). Regulation of the BBB requires cross-talk between endothelial cells, pericytes, smooth muscle cells which invest the penetrating arterioles, the astrocytic foot processes that surround the entire intracerebral vascular network, and neuronal processes which either directly innervate the capillary endothelium or the astrocytic foot processes. This chapter will review the morphology and properties of cerebral endothelium, which is one of the best studied components of the BBB and the neurovascular unit. The biology of brain pericytes is reviewed in Chapter 2 and that of astrocytes in Chapter 3 and the interaction between these cell types as far as is known is discussed in these chapters.

Fig. 1. A modified merged confocal image shows elements of the neurovascular unit in the cerebral cortex. An arteriole consisting of endothelial, smooth muscle, and modified leptomeningeal cell layers is surrounded by astrocytic processes (red). Some of the neuronal processes (green) in the surrounding neuropil are in close proximity to the perivascular astrocytes and the vessel wall.

Morphology and Properties of Brain Endothelial Cells

2. Structural and Molecular Properties

2.1. Caveolae

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Brain endothelial cells have many properties which are similar to those present in nonneural endothelium including the expression of glycoproteins (4, 5), adhesion molecules (6, 7), and integrin receptors (7) which will not be discussed. The principal morphological features which distinguish the endothelial cells of intracerebral vessels from those of nonneural vessels and form the structural basis of the BBB to proteins include reduced density of caveolae and the presence of circumferential tight junctions between endothelial cells. Brain endothelial cells also have increased density of mitochondria. Only these features will be discussed in this chapter. Caveolae were identified by electron microscopy more than 50 years ago (8, 9) in many differentiated cell types including endothelial cells. They are flask-shaped membrane-bound vesicles having a mean diameter of ~70 nm which can open to both the luminal and abluminal plasma membrane through a neck 10–40 nm in diameter. Caveolae are also observed free in the cytoplasm of endothelial cells of most organs where they have a spherical shape (Fig. 2). These vesicles are distinct from clathrin-coated vesicles, which have an electron-dense coat and are involved in receptor-mediated endocytosis. Rapid-freeze followed by deep-etch electron microscopy shows that a striped or striated coat is present at the cytoplasmic surface of caveolae (10). Ultrastructural studies show thin protein barriers anchored in the neck of caveolae,which are named stomatal diaphragms (11–13). The function of these stomatal diaphragms, which are also associated with transendothelial channels

Fig. 2. A segment of arteriolar endothelium from a control rat shows caveolae © in the endothelial and smooth muscle cells. Many of the caveolae show electron-dense deposits representing Ca2+-ATPase localization. X84, 000.

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and vesiculo-vacuolar organelles is unknown. A major component of these diaphragms is the plasmalemmal vesicle protein (PV-1) (14, 15). An association between caveolae and both microtu­ bules  and microfilaments has also been reported in endothelial cells (13). Intracerebral cortical vessels contain a mean of 5 caveolae/mm2 in arteriolar (16) and capillary endothelium (17, 18). Cerebral endothelium contains 14-fold fewer vesicles as compared with endothelium of nonneural vessels such as myocardial capillaries (19). The decreased number of vesicles in cerebral endothelium implies limited transcellular traffic of solutes in steady states. In contrast, capillaries in areas where a BBB is absent such as the subfornicial organ and area postrema (20, 21) are highly permeable and have significantly higher numbers of endothelial caveolae. 2.1.1. Molecular Structure of Caveolae

Studies of nonneural endothelial cells and other cell types have provided information about the molecular structure of caveolae. It is generally accepted that caveolae are lipid rafts (22). The caveolae membrane is enriched in b-d-galactosyl and b-N-acetylglucosaminyl residues in palmitoleic and stearic acids (23) and in cholesterol and sphingolipids (sphingomyelin and glycosphingolipid). The sphingolipids are substrates for synthesis of a second intracellular messenger, the ceramides (24). Cholesterol provides a structural support for caveolae and creates the frame in which many caveolar molecules are inserted. Located in the coat of caveolae are the caveolin (Cav) family of proteins, which comprise three members named Cav-1, 2, and 3. In brain, Cav-1 and 2 are primarily expressed in endothelial cells (25–28), while Cav-3 is expressed in astrocytes (26–28). Cav-2 is tightly coexpressed with Cav-1 in diverse cells including endothelial cells (26, 27), suggesting that both utilize identical transcription regulatory pathways (29). Cav-1, the specific marker and major component of caveolae, is an integral membrane protein (21–24 kDa) having both amino and carboxyl ends exposed on the cytoplasmic aspect of the membrane (30). The two major isoforms of Cav-1 are a and b and brain cells express predominantly the a-isoform (26). There is a link between Cav-1 expression and caveolae formation, as Cav-1-null cells have no caveolae (31) and expression of Cav-1 in cells devoid of Cav-1 results in de novo caveolae formation (32). The precise mechanisms by which caveolins form caveolae are just starting to be unraveled (33, 34). Another molecule having a critical role in caveolae formation is cavin which is also termed polymerase I and transcript release factor. Cavin is abundant at the cytoplasmic face of caveolae (35) being roughly as abundant as Cav-1 in caveolae (36). Cavin expression parallels Cav-1 expression in various tissues and cell lines (36). Cavin is recruited to plasma membrane caveolae domains by Cav-1, and expression of full length cavin seems necessary for caveolae formation in the presence of

Morphology and Properties of Brain Endothelial Cells

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Cav-1 (36). When cavin expression decreases, Cav-1 diffuses in the plasma membrane and becomes internalized into the endolysosomal system where it is degraded, explaining why down-regulation of cavin results in lower expression of Cav-1 (36, 37). Caveolin-1 expression is essential for transcytosis of macromolecules as described in the next section (38). Knockout of the Cav-1 gene results in defects in the uptake and transport of albumin in vivo (39). Cav-1 also acts as a multivalent docking site for recruiting and sequestering signaling molecules through the caveolin-scaffolding domain that recognizes a common sequence motif within caveolin-binding signaling molecules (40). Signaling molecules found in caveolar domains that form complexes with caveolin are: membrane proteins, G protein-coupled receptors, G proteins, nonreceptor tyrosine kinases, nonreceptor Ser/Thr kinases, GTPases, cellular proteins and adaptors, and structural proteins (41) (see Table  1). Cav-1 is also known to regulate endothelial nitric oxide synthase and angiogenesis (42).

Table 1 Proteins associated with endothelial caveolae and their function (41) Protein

Function

Membrane proteins PDGF-R (324)

PDGF receptor

CD36 (63, 325)

Lipoprotein receptor

RAGE (63)

Advanced glycated end products receptor

Gp60 (326)

Albumin receptor

SR-BI (325, 327)

Lipoprotein receptor

Flk-1/KDR (328)

VEGF receptor

Tissue factor pathway inhibitor (329)

Down-regulates the procoagulant activity of tissue factor

Plasmalemmal vesicle protein-1(14)

Component of stomatal diaphragms of caveolae and transendothelial channels

P-glycoprotein (330)

ABC transporter

MMP-1(331)

Matrix metalloproteinase

MMP-2 (332)

Matrix metalloproteinase

Endothelial differentiation gene-1 (EDG-1) receptor (333)

EDG-1 product

uPAR (334)

Urokinase receptor

G protein-coupled receptors B2R (335)

Bradykinin receptor

ETA (336)

Endothelin receptor (continued)

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Table 1  (continued) Protein

Function

G Proteins GaS, Gai1, Gai2,Gbg, (63)

Regulate G protein-coupled receptor activity

Gq (63, 337) Nonreceptor tyrosine kinases Src, Fyn, Yes, Lck, Lyn (63, 324)

Regulation of growth factor response

Tyk2,STAT3(335)

Signal transduction and activator of transcription

Nonreceptor Ser/Thr kinases Raf (338)

Signal transduction of mitogenic signals

MEK (63)

Signal transduction of mitogenic signals

PI-3 kinase (63, 324)

Phosphorylation of phosphatidyl-inositol

PKC a, b (63, 324)

Ser/Thr kinase

Other enzymes eNOS (339, 340)

Production of NO

PLCg (324)

Phospho-lipase

Prostacyclin synthase (341)

Production of prostacyclin (PGI2)

GTPases Ras, Rap1, Rap2 (63)

GTPase

Cellular proteins/adaptors Shc (63)

Regulates growth factor response

Grb2 (63)

Adaptor protein, associates growth factor receptors

Other proteins ER a and b (342)

Estrogen receptors

NCX (343)

Na+/Ca+ exchanger

Ca2+-ATPase (344, 345)

Calcium pump

IP3 receptor-like protein (344, 345)

Involved in calcium influx

Sprouty-1 and -2 (346)

Inhibitor of development-associated RTK signaling

Cationic arginine transporter-1 (347)

Arginine transporter

Structural proteins Actin (63)

Involved in cell motility

Annexin II and IV (63, 345)

Promotes membrane fusion and is involved in exocytosis

Dynamin (337, 348)

Involved in vesicular trafficking

NSF (345)

Involved in vesicle fusion

SNAP, SNARE (345)

Involved in vesicular transport

VAMP-2 (345)

Involved in the targeting/fusion of transported vesicles to their target membranes

Morphology and Properties of Brain Endothelial Cells 2.1.2. Function of Caveolae

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Several lines of research implicate caveolae in the process of vesicular trafficking in transcytosis of proteins (43–46), endocytosis (34, 43, 47), and potocytosis (43). Caveolae regulate a wide variety of signaling molecules (22, 48). In addition, caveolae function in the regulation of cell cholesterol and glycosylphosphatidylinositol (GPI)-linked proteins (49), in cell migration (34), as docking sites for glycolipids (50) and as flow sensors (34, 46). Endothelial caveolae are involved in endocytosis, a process by which the permeant molecules are internalized within endothelial cells or they may be involved in transfer of molecules from blood across the cell to the interstitial fluid or in the reverse direction, a process termed transcytosis (51). Both endocytosis and transcytosis may be receptor-mediated or fluid phase and require ATP and can be inhibited by N-ethylmaleimide (NEM), an inhibitor of membrane fusion (52). Plasma proteins which are essential for many cellular functions are selectively taken up by endothelial cells in caveolae that actively carry cargo across the endothelial cell by receptor-mediated and receptor-independent transcytosis, generally bypassing lysosomes (45, 46, 53). Receptors present in caveolae membranes are involved in receptor-mediated transcytosis of low- and high-density lipoprotein, epidermal growth factor, tumor necrosis factor, albumin, transferrin, melanotransferrin, lactoferrin, ceruloplasmin, transcobalamin, advance glycation end products, leptin, and insulin, all of which are essential in maintaining cell and tissue homeostasis and are therefore referred to as the life receptors (46, 54). Also present are death receptors which are involved in cell apoptosis and include receptors for p75 and interleukin-1 (46). Transcytosis is a multistep process that involves successive caveolae budding and fission from the plasma membrane, translocation across the cell, followed by docking and fusion with the opposite plasma membrane. Caveolae contain the molecular machinery for these processes. Isolated caveolae from lung capillaries demonstrate vesicle-associated membrane protein-2 (VAMP-2) (55), monomeric and trimeric GTPases, annexins II and IV, N-ethyl maleimide (NEF)-sensitive fusion factor (NSF), and its attachment protein – soluble NSF attachment protein (SNAP) and vesicle-associated SNAP receptor (v-SNARE) (56) (see Table 1). These molecules interact in the stages of transcytosis as follows: Caveolae form at the cell surface through ATP-, GTP-, and Mg2+polymerization of Cav-1 and 2, a process stabilized by cholesterol (30). Caveolin oligomers may also interact with glycosphingolipids (57); these protein–protein and protein–lipid interactions are thought to be the driving force for caveolae formation (58). A component of the caveolar fission machinery is the large GTPase, dynamin, which oligomerizes at the neck of caveolae and probably undergoes hydrolysis for fission and release of caveolae, so it becomes free in the cytoplasm (59). Localized at the caveolae neck is intersectin-2 which regulates the GTPase activity of dynamin and

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controls dynamin “pinching” and caveolae-mediated endocytosis (60), while endothelial nitric oxide synthase trafficking inducer (NOSTRIN) functions as an adaptor recruiting dynamin-2 molecules required for vesicle membrane fission (61). Numerous proteins interact with intersectin including SNARE, 25 and 23 kDa synaptosome-associated proteins (SNAP-25 and SNAP-23), actinbinding proteins such as Wiskott-Aldrich syndrome protein, son of sevenless, and a guanine nucleotide exchange factor (62). The transcellular movement of caveolae is facilitated by the association with the actin cytoskeleton-related proteins, such as myosin HC, gelsolin, spectrin, and dystrophin (63). Fusion at the abluminal membrane is aided by NSF, which interacts with SNAPs that can associate with complementary SNAP receptors to form a functional SNARE fusion complex. Prior to fusion of the target and vesicle membrane, v-SNARE (VAMP), the targeting receptor located on the vesicles recognizes and docks with its cognate t-SNARE (syntaxin) on the target membrane (64, 65). Specific docking at the opposite plasma membrane is aided by endothelial VAMP-2 (55), which is localized in caveolae. The evidence thus far favors the hypothesis that caveolae are dynamic vesicular carriers budding off from the plasma membrane to form free transport vesicles that traffic their cargo across cells fusing with specific target molecules on the abluminal plasma membrane as described previously (54, 66–69). Theoretical models of vesicular transport agree in predicting a transport time across endothelium in the order of seconds (38, 70), even against a concentration gradient (38). Caveolae can also fuse to form transendothelial channels extending from the luminal to the abluminal plasma membrane, which allows passage of macromolecules from the blood to tissues or in the reverse direction (53, 68). Such channels have been demonstrated in nonneural vessels in steady states (68), but not in normal cerebral endothelium either by freeze-fracture (71), transmission (72), or high-voltage electron microscopy, which allows examination of 0.25–0.5 mm thick plastic sections (73). Transendothelial channels have been observed in cerebral endothelium following BBB breakdown as discussed in Subheading 5.2.1. 2.2. Endothelial Junctions

Tight junctions are present at the apical end of the interendothelial space being intimately connected to and dependent on the cadherinbased adherens junctions which are located near the basolateral side of the interendothelial space. In brain injury, once breakdown of tight junctions occurs, adherens junctions do not impede the passage of macromolecules.

2.2.1. Tight Junctions

Ultrastructural studies of the tight junctions of brain endothelial cells demonstrate close approximation of the outer leaflets of adjacent plasma membranes forming a pentalaminar structure (74) (Fig.  3), which prevents the passage of tracers such as

Morphology and Properties of Brain Endothelial Cells

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Fig. 3. A segment of arteriolar endothelium shows tight junctions (arrowheads) along the interendothelial space. Caveolae © are associated with the interendothelial space in two locations. ×162, 000.

horseradish peroxidase (HRP) into the brain (75). This suggests that these tight junctions extend circumferentially around the endothelial cells forming a barrier to paracellular passage of small hydrophilic molecules such as sodium, hydrogen, bicarbonate, and other ions, a property referred to as its “gate or barrier” function. Tight junctions also restrict the movement of membrane molecules between the functionally distinct apical and basolateral membrane surfaces, a property referred to as its “fence” function. Freeze-fracture studies demonstrate that the tight junctions of cerebral endothelium consist of 8–12 parallel strands having no discontinuities, which run along the longitudinal axis of the vessel, with numerous lateral anastomotic strands (76). This pattern extends into the postcapillary venules, although in a less complex fashion. In cerebral arteries, tight junctions consist of simple networks of junctional strands, with occasional discontinuities, whereas collecting veins have tight junctional strands which are free-ending and widely discontinuous (76). Freeze-fracture studies also demonstrate that cerebral endothelial tight junctions have a high association with the protoplasmic (P)-face of the membrane leaflet, which is 55% as compared with endothelial cells of nonneural blood vessels, which have a P-face association of only 10% (77). The physiological correlate of tightness in epithelial membranes is transepithelial resistance. Leaky epithelia generally exhibit electrical resistances between 100 and 200 W/cm2, while the electrical resistance across the BBB in vivo is estimated to be approximately 4–8,000 W/cm2 (78, 79). Cultured brain endothelial cells grown in the absence of astrocytes show a 100-fold decrease in electrical resistance to approximately 90 W/cm2 (80),

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while coculture of brain microvascular endothelial with astrocytes increases transendothelial electrical resistance as described in Chapter 3, Subheading 3.2. The electrical resistance of cultured endothelial cells can also be increased to 400–1,000 W/cm2 by using special substrata such as type IV collagen and fibronectin (81). Tight junctions are localized at cholesterol-enriched regions along the plasma membrane associated with Cav-1 (82). Research using a variety of cell types including cerebral endothelial cells demonstrates that tight junctions are composed of an intricate combination of tetraspan and single-span transmembrane and cytoplasmic proteins linked to an actin-based cytoskeleton that allows these junctions to seal the paracellular space while remaining capable of rapid modulation and regulation (see Chapter 5, Fig. 3). The two tetraspan transmembrane proteins are the claudin family of proteins and occludin, while the single-span transmembrane protein is the junction adhesion molecule (JAM) family of proteins. The tetraspan proteins form the paracellular permeability barrier and determine the capacity and the selectivity of the paracellular diffusion pathway. The claudin family consists of 24 members in mice and humans and exhibit distinct expression patterns in tissue and cells (83–86). Claudins are 18–27-kDa tetraspan proteins with a short cytoplasmic N-terminus, two extracellular loops, and a COOHterminal cytoplasmic domain which ends in valine. The latter strongly attracts PDZ (PSD-95/DlgA/ZO-1)-containing proteins such as zonula occludens (ZO)-1-3, PATJ, PALS1, and MUPP1 (87). These interactions are thought to be important for junction assembly. Claudins are considered to be the main structural components of intramembrane strands (88) and recruit occludin to tight junctions (89). Occludin knockout mice are still capable of forming interendothelial tight junctions having normal morphology and barrier function in intestinal epithelial cells (90), while claudin knockout mice are nonviable (91). The extracellular loops of claudins are considered to create aqueous pores that have biophysical properties similar to those of traditional ion channels including ion charge selectivity, permeability dependence on ion concentration, and competition for movement of permeative molecules (92). These channels permit the passive diffusion of mostly cations, but anion passage has also been documented (86, 93). Claudin-5 regulates size-selective diffusion of small molecules since claudin-5 knockout mice show increased paracellular permeability to molecules