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T HE PULMONARY ENDOTHELIUM

The Pulmonary Endothelium: Function in health and disease Edited by Norbert F Voelkel and Sharon Rounds

T HE PULMONARY ENDOTHELIUM

Function in health and disease

Editors

Norbert F Voelkel

Virginia Commonwealth University, Richmond, VA, USA

Sharon Rounds

Alpert Medical School of Brown University, Providence, RI, USA

A John Wiley & Sons, Ltd., Publication

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Library of Congress Cataloguing-in-Publication Data

The pulmonary endothelium / [edited by] Norbert F Voelkel, Sharon Rounds.

A catalogue record for this book is available from the British Library.

Typeset in 9/11pt Times by Laserwords Private Ltd, Chennai, India

Printed in Singapore by Fabulous Printers Pte Ltd.

First Impression 2009

This book is dedicated to our families and to our mentors We particularly acknowledge the contributions of Robert Grover, Ivan

McMurtry, and the late Jack Reeves to our careers.

SECTION I: NORMAL PULMONARY ENDOTHELIUM STRUCTURE, FUNCTION,

1: Development of the Pulmonary Endothelium in Development of the Pulmonary Circulation: Vasculogenesis and Angiogenesis,Margaret A Schwarz and

3: Cadherins and Connexins in Pulmonary Endothelial Function,Kaushik Parthasarathi

4: Pulmonary Endothelial Cell Interactions with the Extracellular Matrix,

Katie L Grinnell and Elizabeth O Harrington 51 5: Pulmonary Endothelial Cell Calcium Signaling and Regulation of Lung Vascular

7: Pulmonary Endothelial Cell Surface Metabolic Functions,Usamah S Kayyali and

8: Cell Biology of Lung Endothelial Permeability,Guochang Hu and

Richard D Minshall 113 9: Lung Endothelial Phenotypes: Insights Derived from the Systematic Study

10: Pulmonary Endothelial Interactions with Leukocytes and Platelets,Rosana Souza

Rodrigues and Guy A Zimmerman 143

viii CONTENTS

11: Mesenchymal–Endothelial Interactions in the Control of Angiogenic, Inflammatory, and Fibrotic Responses in the Pulmonary Circulation,Kurt R Stenmark, Evgenia V.Gerasimovskaya, Neil Davie and Maria Frid . 167 12: Pulmonary Endothelium and Vasomotor Control,Nikki L Jernigan,

Benjimen R Walker and Thomas C Resta 185 13: Pulmonary Endothelial Progenitor Cells,Bernard Th´ebaud and Mervin C Yoder 203 14: Bronchial Vasculature: The Other Pulmonary Circulation,Elizabeth Wagner 217 15: Mapping Protein Expression on Pulmonary Vascular Endothelium,

Kerri A Massey and Jan E Schnitzer . 229

SECTION II: MECHANISMS AND CONSEQUENCES OF PULMONARY ENDOTHELIAL CELL

16: Pulmonary Endothelial Cell Death: Implications for Lung Disease Pathogenesis,

Qing Lu and Sharon Rounds 243 17: Oxidant-Mediated Signaling and Injury in Pulmonary Endothelium,Kenneth E

Chapman, Shampa Chatterjee and Aron B Fisher 261 18: Hypoxia and the Pulmonary Endothelium,Matthew Jankowich, Gaurav Choudhary

20: Effects of Pressure and Flow on the Pulmonary Endothelium,Wolfgang M Kuebler 309 21: Therapeutic Strategies to Limit Lung Endothelial Cell Permeability,

Rachel K Wolfson, Gabriel Lang, Jeff Jacobson and Joe G N Garcia 337 22: Targeted Delivery of Biotherapeutics to the Pulmonary Endothelium,Vladimir R

23: Endothelial Regulation of the Pulmonary Circulation in the Fetus and Newborn,

Yuansheng Gao and J Usha Raj 381 24: Genetic Insights into Endothelial Barrier Regulation in the Acutely Inflamed Lung,

Sumegha Mitra, Daniel Turner Lloveras, Shwu-Fan Ma and Joe G N Garcia . 399

25: Interactions of Pulmonary Endothelial Cells with Immune Cells and Platelets:

Implications for Disease Pathogenesis,Mark R Nicolls, Rasa Tamosiuniene,

Ashok N Babu and Norbert F Voelkel 417

26: Role of the Endothelium in Emphysema: Emphysema – A Lung Microvascular Disease, Norbert F Voelkel and Ramesh Natarajan 437

27: Pulmonary Endothelium and Pulmonary Hypertension,Rubin M Tuder and Serpil C Erzurum . 449

28: Collagen Vascular Diseases and Pulmonary Endothelium,Pradeep R Rai and Carlyne D Cool 461

29: Pulmonary Endothelium in Thromboembolism,Irene M Lang 471

30: Pulmonary Endothelium and Malignancies,Abu-Bakr Al-Mehdi . 485

Epilogue,Norbert F Voelkel . 491

Index . 495

Department of Biochemistry and Molecular Biology, Center for Lung Biology, College

of Medicine, University of South Alabama, Mobile, AL 36688, USA

Department of Pharmacology and Center for Lung and Vascular Biology, University

of Illinois College of Medicine, Chicago, IL 60612, USA

JEFF JACOBSON

Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, USA

LIST OF CONTRIBUTORS xiii MATTHEW JANKOWICH

Alpert Medical School of Brown University, Vascular Research Laboratory, dence VA Medical Center, Providence, RI 02908, USA

Provi-NIKKI L JERNIGAN

Vascular Physiology Group, Department of Cell Biology and Physiology, University

of New Mexico Health Sciences Center, Albuquerque, NM, USA

DANIEL TURNER LLOVERAS

Pritzker School of Medicine, Department of Medicine, Section of Pulmonary/Critical Care Medicine, University of Chicago, Chicago, IL 60637, USA

Vascular Physiology Group, Department of Cell Biology and Physiology, University

of New Mexico Health Sciences Center, Albuquerque, NM, USA

LIST OF CONTRIBUTORS xv ROSANA SOUZA RODRIGUES

Department of Radiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

NORBERT VOELKEL

The E Raymond Fenton Professor of Pulmonary Research, Director, Victoria Johnson Center for Pulmonary Obstructive Disease Research, Pulmonary and Critical Care Medicine Division, Virginia Commonwealth University, Richmond, VA 23298, USA

ELIZABETH WAGNER

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224, USA

BENJIMEN R WALKER

Vascular Physiology Group, Department of Cell Biology and Physiology, University

of New Mexico Health Sciences Center, Albuquerque, NM, USA

1Vascular Research Laboratory, Providence VA Medical Center, Alpert Medical School of Brown

University, Providence, RI, USA

2Victoria Johnson Center for Pulmonary Obstructive Disease Research, Pulmonary and Critical Care

Medicine Division, Virginia Commonwealth University, Richmond, VA, USA

Over the past 40 years there has been an explosion of

new knowledge regarding normal and abnormal

func-tion of vascular endothelium In the past, endothelium

was regarded as a passive lining of blood vessels with

organ-specific variability with regard to its role in

tion of blood or in maintenance of minimal fluid

filtra-tion As the nonrespiratory functions of the lung became

recognized, the importance of the endothelium became

evident In his review on this topic in 1969, Fishman

stated with prescience “It is clear from the

observa-tions and speculaobserva-tions above that the degree to which the

pleuripotential [sic] endothelial cells actually fulfill their

potential promises to be a rewarding line of investigation”

[1] Indeed, with the advent of recognition of metabolic

functions of endothelium, it became clear that the

en-dothelium is critical to maintenance of a thrombosis-free

surface, to interactions with circulating blood cells, and

to modulation of vasomotor tone This Introduction and

this volume are not intended to enumerate all of the

in-vestigators and their contributions to the understanding

of lung endothelial pathobiology, but to describe

high-lights in the field and to describe the current state of

understanding

The lung endothelium is now recognized to have a

number of unique functional attributes that are due to

its central location in the circulation The entire cardiac

output passes through the lung with every heartbeat

Furthermore, the lung endothelium has a vast surface

area, estimated to be 120 m2 Thus, lung endothelium

is uniquely positioned to interact with circulating cells

and vasoactive mediators Indeed, it is now clear that the

pathogenesis of many lung diseases, such as acute lung

injury, is related to this important attribute

Another unique feature of the lung endothelium is the

need for the lung to maintain a relatively dry

intersti-tial and alveolar gas space to facilitate gas exchange.The anatomic features of lung endothelium are critical tofluid and protein filtration, and crucial for normal lungfunction The ultrastructural features of the pulmonarycapillary endothelium important in maintenance of nor-mal lung vascular permeability [2] and the effects ofinjury on endothelium have been elegantly described [3].There has also been an enormous increase in understand-ing of the cell biology of lung endothelial permeabilityand the effects of injury on signaling mechanisms, such

as increased permeability caused by thrombin [4].The study of the lung endothelium originally used thestudy of the metabolism of circulating substances, such

as angiotensin I [5], 5-hydroxytryptamine (serotonin) [6],and eicosanoids [7], using passage through isolated per-fused lungs [8] Similarly, isolated perfused lungs wereused to assess perturbation of endothelial permeability[9] The advent of techniques for isolation and culture ofendothelial cells (EC)s from umbilical veins [10, 11], themain pulmonary artery [12], and pulmonary microves-sels [13–15] has allowed the study of endothelium alone,without confounding factors related to distribution ofperfusate Correlation of results using cultured ECs andintact lungs was an important advance in the field [16]

In addition, the availability of cultured endothelium hasallowed elucidation of the interactions of ECs with bloodcells and platelets More recently, with the advent ofanimal models of disease and genetically manipulatedmodels, emphasis has shifted to the study of endothelium

of intact lungs

Recent research has made clear that the lung ECsare heterogeneous in calcium handling, permeability, andproliferative potential with differences between endothe-lium of conduit vessels and the microcirculation, as de-scribed in Chapters 5 and 9 of this volume Furthermore,

the bronchial and pulmonary circulations differ in their

physiology and responses to disease, as discussed in

Chapter 14 It is now apparent that the lung

endothe-lium is not a static organ, but is capable of regeneration

and repopulation via resident and circulating progenitor

cells, as described in Chapter 13

The pulmonary circulation, unlike the systemic

cir-culation, is a low-pressure, high-volume circulation that

responds to hypoxia with vasoconstriction The lung

en-dothelium is critical to maintenance of normal lung

vas-cular tone and modulation of hypoxic vasoconstriction,

reviewed in Chapter 12 In addition, the pulmonary

circu-lation responds to alveolar hypoxia with vascular

remod-eling and sustained pulmonary hypertension The lung

endothelium again is key in modulation of pulmonary

vascular remodeling, as discussed in Chapters 11 and 27

The most recent group of very exciting advances is

the growing recognition that the lung endothelium plays

an important role in the pathogenesis of lung diseases

and this work is highlighted in this volume in Chapters

23–30 It has become increasingly clear that many lung

diseases are directly due to or complicated by pulmonary

EC dysfunction

This volume is a group of essays that describe the

state-of-the-art knowledge of lung endothelium The

vol-ume is divided into three sections The first section

de-scribes the Normal Pulmonary Endothelium, including

development, structure, cell biology, signaling, functions,

heterogeneity, interactions with circulating cells and

mes-enchymal cells, and the endothelium of the bronchial

circulation The second section of the volume deals with

Mechanisms and Consequences of Pulmonary

Endothe-lial Cell Injury, ranging from effects on ECs to organ

injury, including protection against lung permeability

and drug targeting to pulmonary endothelium The third

section of the volume focuses on the Pulmonary

En-dothelium in Disease Although not a diseased state, this

includes the transition from the fetal to the newborn

lung Throughout the volume, it will be evident that these

sections are somewhat arbitrary since insights into normal

function inevitably enhance understanding of

pathophys-iology and vice versa

We are grateful to the authors who have contributed

outstanding chapters that reflect both their work and

overviews of the field We are also grateful to our

colleagues and spouses for their support of this effort

Finally, we thank our publishers, especially Fiona Woods

of John Wiley & Sons, Ltd, who has patiently and firmly

encouraged the completion of this work

References

1 Heinemann, H.O and Fishman, A.P (1969)

Non-respiratory functions of mammalian lung Physical

Review , 49, 1–47.

2 Schneeberger-Keeley, E.E and Karnovsky, M.J.(1968) The ultrastructural basis of alveolar-capillarymembrane permeability to peroxidase used as a

tracer Journal of Cell Biology, 37, 781–93.

3 Bachofen, M and Weibel, E.R (1977) Alterations

of the gas exchange apparatus in adult ratory insufficiency associated with septicemia

respi-American Review of Respiratory Disease, 116,

589–615

4 Mehta, D and Malik, A.B (2006) Signaling

mecha-nisms regulating endothelial permeability Physical

Review , 86, 279–367.

5 Fanburg, B.L and Glazier, J.B (1973) Conversion

of angiotensin 1 to angiotensin 2 in the isolated

perfused dog lung Journal of Applied Physiology,

35, 325–31.

6 Block, E.R and Fisher, A.B (1977) Depression ofserotonin clearance by rate lungs during oxygen

exposure Journal of Applied Physiology:

Respira-tory, Environmental and Exercise Physiology, 42,

8 Dawson, C.A., Bongard, R.D., Rickaby, D.A et al.

(1989) Effect of transit time on metabolism of a

pulmonary endothelial enzyme substrate American Journal of Physiology: Heart and Circulatory Phys-

morphologic and immunologic criteria Journal of

INTRODUCTION xix

12 Ryan, U.S., Clements, E., Habliston, D., and Ryan,

J.W (1978) Isolation and culture of pulmonary

artery endothelial cells Tissue and Cell , 10,

535–54

13 Ryan, U.S., White, L.A., Lopez, M., and Ryan, J.W

(1982) Use of microcarriers to isolate and culture

pulmonary microvascular endothelium Tissue and

Cell , 14, 597–606.

14 Alvarez, D.F., Huang, L., King, J.A et al (2008)

Lung microvascular endothelium is enriched with

progenitor cells with vasculogenic capacity

Amer-ican Journal of Physiology: Lung Cellular and

Molecular Physiology, 294, L419–30.

15 Masri, F.A., Xu, W., Comhair, S.A.A et al (2007)

Hyperproliferative apoptosis-resistant endothelial

cells in idiopathic pulmonary hypertension ican Journal of Physiology: Lung Cellular and

endothelial cell

fibrous connective tissueexternal elastic tissuesmooth muscle (tunica media)internal elastic tissueendothelium (tunica intima)

Plate 1.2 Fundamental architecture of blood vessels

The Pulmonary Endothelium: Function in health and disease Edited by Norbert F Voelkel and Sharon Rounds

epithelial VEGF gradients, the vasculogenic pools, and angiogenic extensions from the growing lung plexus.

photo- excitation

20 µm

Gray Levels

170850

Plate 3.4 GJ-dependent responses in lung microvessels Reproduced from Parthasarathi et al (2006), The Journal of

Clinical Investigation, 116, 2193–200 by permission of the American Society for Clinical Investigation.

AWAdv

(a) (b) (c)

+BQ788+BQ123

Plate 11.7 Fluorescence microscopy showing that cord-like networks, formed in hypoxic VVEC-AdvFBs Matrigelco-cultures (a), were markedly attenuated when cells were incubated with either the ETAreceptor antagonist BQ123 (b)

or the ETBreceptor antagonist BQ788 (c)

?

Endothelial CellCluster

MaturaDifferentialedEndothelium

Plate 13.1 Model of an EPC hierarchy based on the proliferative and clonogenic potential of discrete populations ofprogenitor cells

150

100

50 150

ALI/VILI Candidate Genes

Dahl Salt Sensitive (SS) Resistant to VALI

AKT GSK3 mTOR BAD Apoptosis

Blood Coagulation

Inflammatory Response

Regulation Cell Proliferation

Cytoskeleton Chemotaxis

Immune Response

PI3K

VILI Genes

Barrier Regulation

Protein Synthesis

Orthologous Gene

Expression

Consomic Rodent Models

Approach with Expression Profiling

Signaling Pathway Analysis

Plate 24.1 Representative novel approaches to identify ALI-implicated genes

(a)

(b)

Vessel

Plate 26.1 (a) Human lung tissue sections Reproduced from Nana-Sinkam et al (2007), American Journal of

Respiratory and Critical Care Medicine, 175, 676–85 with the permission of the American Thoracic Society (b) Terminal

deoxynucleotidyl transferase biotin-dUTP nick end-labeling staining of a lung vessel in a human emphysema lung section

demonstrates apoptotic ECs within the vessel EC monolayer (arrows) Reproduced from Kasahara et al (2001) American

Journal of Respiratory and Critical Care Medicine, 163, 737–44 with the permission of the American Thoracic Society.

(d) (e)

Plate 27.1 Plexiform lesions occurring along two branches of medium-sized pulmonary arteries (arrows) Reproduced

from Cool et al (1999) American Journal of Pathology, 155, 411–19, by permission of the American Society for

Investigative Pathology

(c)

Plate 27.2 Expression of HIF-1α in a plexiform lesion (a) and in a concentric lesion (b), and of HIF-1β in a plexiform

lesion (c) Reproduced from Tuder et al (2001) Journal of Pathology, 195, 367–74 with permission from John Wiley

& Sons, Ltd

Plate 27.3 Cellular localization of phospho-STAT-3 by immunohistochemical staining in IPAH lung Reproduced from

Masri et al (2007), American Journal of Physiology: Lung Cellular and Molecular Physiology, 293, L548–54, with

permission from The American Physiological Society

Plate 28.1 Pulmonary artery from a patient with diffuse scleroderma/SSc showing a marked thickening of the adventitialcollagen (double arrow)

appearance of the ECs.

Plate 28.3 (a) Pulmonary artery obliterated by a concentric, “onionskinning,” proliferation of ECs, highlighted byimmunohistochemical stain for ECs (Factor VIII-related antigen) (b) Dilatation lesion at the distal end of a plexiformlesion Immunohistochemical stain for EC marker, CD31

Plate 28.4 Bifurcating pulmonary artery from a patient with CREST and severe PAH.

Plate 29.4 Characterization of cells in a vena cava thrombus of the mouse

Plate 29.5 Representative histological section of a chronic pulmonary embolus, illustrating an area with in situ

thrombosis

control Anti-SMC

Plate 29.7 Trichrome stain of a histological section of a thrombus from a patient with chronic thromboembolicpulmonary hypertension.

Plate 29.8 Characterization of cells in a vena cava thrombus of the mouse

Development of the Pulmonary

Endothelium in Development of the

Pulmonary Circulation: Vasculogenesis

and Angiogenesis

1Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas,

Dallas, TX, USA

2Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas,

Dallas, TX, USA

INTRODUCTION

Role of the Pulmonary Vasculature

The cardiovascular system, comprised of the heart and

blood vessels, is the first functional organ formed during

embryogenesis in higher vertebrates In the mouse, the

heart and first vessels become functional as early as 8

days following fertilization, while in humans the

cardio-vascular system forms after approximately 3 weeks of

development Cardiovascular function is essential to the

survival of higher organisms, because every cell requires

nutrition, gas exchange, and elimination of wastes via

blood vessels The primary site of gas exchange is

the vascular/alveolar interface, located deep within the

lung Once blood is oxygenated in the lung, pumping

of the blood by the heart disperses oxygen-rich blood

throughout the body, where exchange of gas within

tissues occurs via capillary beds Then, oxygen-depleted,

carbon dioxide-rich blood is returned to the lungs via

the vena cava, for the respiratory/circulatory cycle

to begin anew Despite decades of research into the

biology of this vascular/pulmonary interface, little is

known about how the pulmonary vasculature ensures

its proper coordinated growth and intimate

develop-ment along the tree-like epithelium of the developing

lung

Vascular Development Overview

Morphogenesis of the embryonic vascular systembegins with the emergence of angioblasts, or endothelialprogenitor cells, which are initially scattered within themesoderm prior to their incorporation into patent vessels[1] Angioblasts are fibroblast-like, mesodermal cellscapable of migrating, recognizing other angioblasts,adhering, and organizing into vascular structures Once

an angioblast is recruited into forming a vascular “tube,”

or vessel, it differentiates into a bona fide differentiatedendothelial cell (EC) The defining cell type of the estab-lished cardiovascular system is thus the EC, which formsthe seamless lining of the entire circulatory system Asthe vasculature develops, the initial circulatory system iscomposed of a rather homogeneous system of primitivevessels, or “plexus.” However, as the embryo develops,this plexus reshapes and remodels into a hierarchical net-work of large and small vessels In large vessels, such asthe major arteries and veins, the endothelial inner liningbecomes insulated by thick layers of extracellular matrix(ECM) components and smooth muscle In capillarybeds, where vessels taper to very narrow diameters, andgases and nutrients are actively exchanged, the endothe-lium is relatively more “naked” and in immediate contactwith surrounding tissues Thus, development of the vas-

The Pulmonary Endothelium: Function in health and disease Edited by Norbert F Voelkel and Sharon Rounds

cular system is a step-wise series of dynamic cellular

activities, which together shape individual blood vessels,

thereby ensuring proper distribution of oxygen-rich

blood throughout the body Interestingly, most key steps

in specification and differentiation of vascular cell types

are driven by the molecular interaction of vascular

en-dothelial growth factor (VEGF) with its receptor vascular

endothelial growth factor receptor VEGFR-2, which is

expressed in vascular ECs In this chapter, we will review

the basic steps during systemic and pulmonary vessel

development, since they are driven by many analogous

mechanisms, and we will present new ideas regarding

the molecular basis of their coordinated growth

ONTOGENY OF VASCULAR CELLS

Endothelial Origin

To fully understand vascular development, it is essential

to know where exactly endothelial precursors come from

Although their exact cell of origin has long remained

elusive, angioblasts are known to differentiate exclusively

from the mesoderm [2, 3] In addition, it has been

demonstrated that angioblasts arise in both extra- and

intra-embryonic mesoderm, with their extra-embryonic

emergence in the yolk sac preceding their differentiation

in embryonic tissues In mouse, the first extra-embryonic

angioblasts can be detected as early as embryonic day (E)

6.5, while those in the embryo proper can be identified

later, around E7.0 [4–6] The first angioblasts identified

in the yolk sac can be found within local proliferative

foci of extra-embryonic mesoderm These aggregations

of angioblasts progressively take a more definitive shape,

either as angioblast “cords” (linear aggregates) or blood

islands (see following section) [5, 6] In all vertebrates

examined, these primitive vascular structures precede the

formation of a functional and continuous vasculature

Blood Islands and Hemangioblasts

As mentioned in the previous section, some of the earliest

angioblasts identified in vertebrates are those in or near

structures called “blood islands” [5, 7] In mouse, blood

islands are scattered in a ring around the distal yolk sac

mesoderm [8–10] In frog and fish, on the other hand,

a single blood island is found on the ventral aspect of

the gut Blood islands have been described as

“mesoder-mal cell aggregates,” where inner cells consist of blood

or hematopoietic stem cells and outer cells comprise a

mantle of angioblasts [5] Thought to represent

transi-tional structures, blood islands have been shown to grow

and fuse, creating a continuous network of blood filled

vessels [6, 11, 12] However recent work calls into

ques-tion this “blood island fusion” mechanism of vascular

development, and suggests instead that embryonic sels are more likely to derive from ECs migrating andenveloping, or “capturing,” hematopoietic precursors, asthey generate a continuous vasculature [5] Regardless ofthe exact dynamics, blood islands have been observedfor over a century and are a hallmark of the primitivevertebrate yolk sac vasculature

ves-The close spatial and temporal association ofhematopoietic and EC development in the yolk sacblood islands led to the idea that both lineages originatedfrom common precursor called the “hemangioblast” [1,13–16] This possibility is supported by the observationthat vessel and blood progenitors express many commonmarkers and mutation of a number of genes affects bothlineages [11, 17] For decades, evidence has accumulatedthat supports the existence of a hemangioblast [18–20].However, the isolation of a truly bipotential cell in theembryo, with the capacity to give rise exclusively toboth EC and hematopoietic cell types, has yet to beconclusively shown Recent experiments demonstratethat most intra-embryonic ECs do not emerge from bloodislands, and in addition, few blood and ECs actuallyarise from common progenitors [21–23] Therefore,the question remains open as to the true nature of thehemangioblast, the breadth of its potential to give rise todifferent cell types, and its actual frequency within theearly vertebrate embryo

The Endothelial Cell

The fundamental building unit of the blood vessel is the

EC Together, blood vessels of an adult human consist

of approximately 1× 1013ECs, which stitch together toform the hierarchical network of vessels that carry bloodthroughout the body [24] One interesting question thatarises is exactly how does one define the EC? Only twoshared characteristics have been identified that can be ap-plied to all ECs [25] The first is anatomical, in that ECsadhere to one another and form the seamless inner lin-ing of all blood vessels The second is functional, in thatECs create a selectively permeable and active interface,between blood and tissues, which controls the passage

of nutrients, gases, and immune cells Surprisingly, yond these two traits, no single definition can be appliedglobally to all ECs Blood vessels are strikingly differentfrom one tissue to the next It has been said that there are

be-as many different types of ECs be-as there are tissues [26]

In the last decade, ECs have been shown to be extremelyheterogeneous in their transcriptional profile, structuralfeatures, and regionalized functions [27–29] Therefore,perhaps a more apt definition of ECs is that they can gen-erally be defined as the cells that line the lumen of bloodvessels, but display a variable nature that is strikinglyheterogeneous, dynamic, and plastic

ONTOGENY OF THE VASCULATURE 5

ONTOGENY OF THE VASCULATURE

Cellular Mechanisms of Blood Vessel Formation

Blood vessel development occurs via two principal and

distinct cellular mechanisms, referred to as

vasculoge-nesis and angiogevasculoge-nesis (Figure 1.1) [15, 30, 31–34]

The initial primitive vascular plexus emerges via

vas-culogenesis, which describes the de novo formation of

blood vessels from individual angioblasts Angiogenesis,

in contrast, describes the growth and remodeling of the

existing primitive vasculature, and occurs during normal

growth of embryonic organs and tissues Both

vasculo-genesis and angiovasculo-genesis strictly refer to “the vasculo-genesis

of blood vessels”; however, they have been used to

de-scribe very different cellular mechanisms of blood vessel

formation

Vasculogenesis

Vasculogenesis refers to the formation of blood vessels

via the clustering and organization of individual

an-gioblasts into linear aggregates, or “cords,” followed by

the formation of a patent lumen (Figure 1.1a) [15, 30, 35,36] In addition, the term has also been used to describethe fusion of blood islands into blood-filled tubes withinthe yolk sac Vasculogenesis is known to be the primarymechanism by which the first embryonic vessels form [2,36] This includes the primordia of most primitive bloodvessels, including the dorsal aortae and the endocardium,

as well as the relatively homogeneous capillary networkfound in tissues such as the yolk sac Vasculogenesis istherefore a term that describes a step-wise developmentalprocess, which includes angioblast migration, prolifera-tion, adhesion, morphogenesis, differentiation, and matu-ration into ECs Coalescence of these individual vascularprogenitors ultimately leads to the formation of a con-tinuous network of vessels, which circulation depends

on “Vasculogenesis” and “neovascularization” are both

terms that refer to this de novo formation of blood

ves-sels, and are often used interchangeably Two types ofvasculogenesis have been described, type 1 and type

2, with the distinction being based on the location ofangioblast emergence relative to the location of vesselformation In type 1, angioblasts aggregate into cords, at

plus Angiogenesis

(b) Sprouting Angiogenesis

(c) Angiogenic Remodeling

Figure 1.1 Schematic illustrating the different mechanisms of blood vessel formation (a) Vasculogenesis is the de novo

formation of vessels via aggregation of angioblasts within the mesoderm (b) Sprouting angiogenesis is the formation andextension of new sprouts from pre-existing vessels (c) Angiogenic remodeling is the reorganization and shape change

of vessels within an existing vascular plexus (d) In many tissues, including lung, vasculogenesis and angiogenesis arecoordinated to create vascular beds within developing organs and tissues

the same location where they emerge in the mesoderm.

In type 2, angioblasts appear in the mesoderm, but then

actively migrate to a different location, where they then

coalesce into vessels During embryonic vascular

devel-opment, dorsal aortae formation in mouse occurs by

vas-culogenesis type 1 [37], while the formation of a single

dorsal aorta in frog entails vasculogenesis type 2 [38, 39]

Tubulogenesis

Central to the concept of vasculogenesis is the concept

of endothelial tubulogenesis Morphogenesis of a

vas-cular “tube,” from a “cord” of angioblasts or within

a growing angiogenic sprout, occurs via tubulogenesis

Tubulogenesis has been described as occurring by two

distinct mechanisms In the first mechanism, the vascular

lumen forms by the alignment and fusion of “intracellular

spaces,” such as large vacuoles [40, 41] Classical

obser-vations in the avian embryo suggest this first mechanism,

where a lumen can be shown to form from the fusion

and expansion of intracellular vacuoles into a long

con-tinuous space across many cells, at the center of a cord

[40–45] Alternatively, the lumen can be generated by

the enlargement of an “extracellular space” located

be-tween adjacent angioblasts [46] The latter mechanism

for vascular “tube” formation primarily involves cellular

rearrangements that drive the transformation of a solid

cord of cells, into a patent cylinder Based on zebrafish

observations [46], it might be predicted that vacuole

fusion-based tubulogenesis is likely to be predominantly

used in angiogenic sprouting as discussed below, whereas

rearrangement-based tubulogenesis is likely to occur

pri-marily during vasculogenesis

Angiogenesis

Following the formation of the initial primitive

vas-cular plexus via vasculogenesis, the simple circulatory

system is then elaborated and extended via

angiogene-sis Two fundamentally distinct angiogenic mechanisms

have been identified: “sprouting angiogenesis” and

“an-giogenic remodeling.” Sprouting angiogenesis is defined

as the sprouting and extension of new vessels from

pre-existing vessels Quiescent cells within the walls of

vessels proliferate, branch, and extend new sprouts into

avascular tissues Angiogenic remodeling encompasses

the multiple gross changes that pre-existing vessels can

undergo in their basic size or pattern, including the

split-ting or fusion of the vessel and the enlargement or

shrink-ing of vessel diameter [47–49] Often these changes in

vessel size or shape occur in response to hemodynamic

forces Here, we describe the general features

distinguish-ing each type of angiogenesis

Sprouting AngiogenesisSprouting angiogenesis involves sprouting of new cap-illaries from the walls of pre-existing blood vessels(Figure 1.1b) Quiescent cells at a specific point alongthe vessel wall initiate a cascade of targeted cellularactivities, all aimed at building an entirely new vesselbranch from a pre-existing parent vessel To create a newsprout, proteolytic degradation of the ECM surroundingthe parent vessel is coordinated with proliferation of thesprouting ECs Together these cellular activities generate

a new growing vascular branch, which will eventuallyfuse with the wall of an adjacent vessel

Cells at the distal tip of extending angiogenic sprouts,termed “tip” cells, have attracted recent attention Newcapillary sprouts grow into the interstitium by the ame-boid migration of distal tip ECs These invade surround-ing avascular tissue, migrate as the sprout extends, fusewith the endothelium of an adjacent vessel, and open up

a new connecting lumen [14] Interestingly, the growth

of new sprouts is not believed to occur by proliferation ofthe tip cells As the angiogenic sprouts extend, it is withinthe growing stalk that new cells are added by mitotic pro-liferation of pre-existing ECs [50] Classical observations

of neural angiogenesis demonstrated that ECs located atthe tip of sprouts exhibited a number of distinctive “fili-form” processes, hypothesized to function in seeking outand fusing with other growing vessels [51] More recentstudies on endothelial tip cell filopodia in growing retinalvessels have shown that filopodia are the primary target

of VEGF signaling and function to drive vessel growthand extension [52, 53]

Remodeling Angiogenesis

Another angiogenic process that generates basic genetic changes in the vascular network architecture is

morpho-“remodeling angiogenesis,” or “angiogenic remodeling.”

In this angiogenic process, pre-existing vessels change inshape, size, and fundamental organization (Figure 1.1c).Generally, these changes involve a wide range of cellu-lar modifications that dynamically alter blood vessel size

or architecture During remodeling, vessels of an initialembryonic plexus either enlarge or regress during de-velopment, accommodating the coordinated growth anddifferentiation of other tissues Once the vascular system

is mature, the vascular network becomes relatively ble and undergoes angiogenic remodeling only in selecttissues, such as in female reproductive organs, woundhealing, or during pathological processes (e.g., tumorgrowth)

sta-A dramatic example of angiogenic remodeling volves the primary capillary plexus of the early murineyolk sac Initially, this plexus presents as a relatively

in-ARTERIAL VERSUS VENOUS DIFFERENTIATION 7

homogeneous network of vessels, resembling a

fisher-man’s net, with most vessels being of equal size, length,

and similar appearance However, this primitive plexus is

rapidly remodeled and modified into the familiar

hierar-chical, tree-like array of larger and smaller blood vessels

These transformations occur via “angiogenic remodeling”

[31, 54] Angiogenic remodeling remains poorly

under-stood, despite the fact many mouse mutants display clear

failure of vascular remodeling

A wide variety of cellular mechanisms underlie

angio-genic remodeling, causing either an increase or decrease

in vessel density Here, we describe intussusception,

re-gression, and pruning Intussusception is the process of

splitting and reorganizing pre-existing vessels, resulting

in the expansion of a capillary network [55, 56]

Dur-ing intussusception, proliferation of ECs within a vessel

results in the formation of a large lumen that is

subse-quently split by intervening endothelial walls (thus

re-sulting in the splitting of one vessel into two) Another

mechanism of vascular remodeling, which in contrast

decreases capillary density, involves endothelial

regres-sion [57] Key steps in vessel regresregres-sion include changes

in EC shape, lumen narrowing, increased vacuolation,

cessation of blood flow, detachment from the basement

membrane, and cell death Regression of vessels often

oc-curs as a result of either a reduction of blood flow,

cessa-tion of VEGF-mediated maintenance, or other genetically

determined processes, such as changes in expression of

angiogenic cues in surrounding tissues Yet another type

of vascular remodeling, which also decreases vessel

den-sity and does not involve cell death, has been termed

“pruning,” as it resembles the process of thinning out

ex-cess branches on a tree [31] Pruning was first observed

in the embryonic retinal vasculature and involves the

re-gression of redundant, parallel channels [58] In these

vessels, blood flow ceases, their lumens collapse and ECs

retract out of the regressing vessel In all cases of

angio-genic remodeling described above, the principal goal is

to fine tune the vasculature so that it perfuses tissues at

the required density, satisfying local oxygen demands,

by trimming excessive, unneeded vessels or reorganizing

vessels to meet physiological demands

Vasculogenesis and Angiogenesis within Organs

Vascularization of most developing embryonic organs has

long been thought to occur primarily via angiogenic

in-vasion of vessels This was a sensible supposition, given

that growing organs appeared to be vascularized by

in-growth of vessels that originated and sprouted from the

pre-existing primary vascular plexus However, improved

technology for visualization of the vasculature and its

precursors, using newly identified molecular markers and

new vascular reporters, has revealed that most organs

de-velop at least part of their vasculature via in situ

aggrega-tion of local mesenchymal angioblasts or vasculogenesis[34] This holds true for the growing vasculature of thelung, liver, stomach, spleen, pancreas, intestine, and kid-ney [32, 59–63] During embryonic development of theseorgans, it is known that angiogenic sprouting from exist-ing vessels also contributes to maintenance and extension

of the primitive organ vasculature [34] New observationshave demonstrated that peripheral vasculogenic vesselsoften fuse with invading angiogenic vessels [64] Thus, itseems likely that building a continuous vasculature withinmost organs is a coordinated joining of both vasculogenicbeds with angiogenic ingrowth of sprouting vessels

ARTERIAL VERSUS VENOUS DIFFERENTIATION

Once blood flow begins within the circulatory system,the immature vascular plexus becomes segregated intorecognizable arteries and veins (Figure 1.2) Vessels can

be categorized as either veins or arteries by a number ofparameters, including the direction of blood flow withintheir lumens, anatomical and functional differences, aswell as by the expression of several markers For instance,the expression of ephrin B2 (Efnb2) ligand is enriched

in arteries, while expression of the B4 ephrin receptor(EphB4) is enriched in veins In addition, a variety ofother markers are specific for arteries, including Dll4 [65,66], Jag1 [67], Notch1 [68], Hey1 and Hey2 [69], activinreceptor-like kinase 1 [70], and EPAS1/hypoxia-induciblefactor (HIF) [71]

The mechanisms underlying the specification of terial and venous cell fate are largely unknown Pre-viously, circulatory dynamics were thought to be thedriving cause of arteries and veins developing into struc-turally and functionally different vessels However, grow-ing evidence points to a genetic program underlying thisfundamental distinction Indeed, labeling experiments inzebrafish suggest that arterial and venous EC fate may bedetermined before the formation of blood vessels [72].Similarly, work in chicks has demonstrated that segrega-tion of arterial and venous markers has already occurred

ar-in subpopulations of blood islands long before vesselformation [73] Therefore, growing evidence points tohard-wired genetic cues specifying arteriovenous cell fateextremely early during vascular development

Interestingly though, it also seems likely that ent vascular beds experience artery/vein specification atdifferent times For instance, arteriovenous markers incertain organs, such as myocardium [74] and pancreas(Cleaver, unpublished), appear to acquire their identi-ties much later during development In addition, it is

Figure 1.2 Fundamental architecture of blood vessels Capillary beds perfuse tissues Capillaries are small calibervessels, the lumen often forming from single ECs Capillaries are largely devoid of supportive cells, except for sparsecoverage by pericytes Capillaries are connected in a hierarchical fashion to larger arterioles and venules, which in turnconnect to arteries and veins Arteries and veins are insulated by thick layers of elastic, smooth muscle and fibroustissues A color version of this figure appears in the plate section of this volume.

known that arteriovenous cell fate is highly plastic and

re-versible In grafting experiments in chicks, vascular ECs

were shown to be plastic with respect to their

arteriove-nous fate [75] In these experiments, fragments of arteries

were heterotopically transplanted to different embryonic

sites Strikingly, cells from the grafted arteries would

quickly colonize either host arteries or veins When they

colonized veins, arterial ECs turned off arterial markers

and upregulated venous markers Thus, EC fate remains

plastic with respect to arteriovenous differentiation, at

least for a period of time during early development

KEY MOLECULES IN VASCULAR

DEVELOPMENT

VEGF [76, 77], and its receptors VEGFR-1 (also called

Flt-1) and VEGFR-2 (also called KDR or Flk-1) [78]

have long been known to be critical regulators of

en-dothelial differentiation, as well as blood vessel formation

and morphogenesis [79] VEGF-A is essential for proper

vessel formation and selective expression of VEGF-A

isoforms (murine 120, 164, 188; human 121, 145, 165,

189, 206) drives different aspects of vessel formation

in many different organs, including the lung [80] Here,

we introduce the principal vascular developmental factorsand outline their roles in vessel formation

VEGF-A and its Isoforms

The VEGF family of growth factors consists of VEGF-A,

B, C, D, and E, and placental growth factor (PlGF).All family members regulate at least some aspect of ECproliferation, migration, and/or survival [79, 81] Genetargeting demonstrates that VEGF-A plays an essentialrole in early vessel development VEGF-A expression isdynamic throughout embryonic development and is oftenexpressed in tissues immediately adjacent to developingblood vessels [38, 77, 82, 83] VEGF-mediated signalingdrives both vessel formation by vasculogenesis, as well asangiogenic invasion of developing tissues Mice lacking

a single VEGF allele die early during embryogenesis(around E10.5) These VEGF-null embryos show a range

of vascular defects, including severe abnormalities in

EC differentiation, sprouting angiogenesis, vessel lumen