Series Editors: D. Neil Granger & Joey Granger
`
`COLLOQUIUMSERIES IN
`INTEGRATED SYSTEMS PHYSIOLOGY:
`FROM MOLECULE TO FUNCTION
`
`Angiogenesis
`
`Thomas H.Adair
`Jean-Pierre Montani
`
`LIFE SCIENCES
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`WS MORGAN & CLAYPOOL LIFE SCIENCES
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`Angiogenesis
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`
`
`Editors
`D. Neil Granger, Louisiana State University Health Sciences Center-Shreveport
`Joey P. Granger, University ofMississippi Medical Center
`Physiology is a scientific discipline devoted to understanding the functions of the body. It addresses
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`Copyright © 2011 by Morgan & Claypool Life Sciences
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`in printed reviews, without the prior permission of the publisher.
`
`Angiogenesis
`Thomas H. Adair and Jean-Pierre Montani
`www.morganclaypool.com
`
`ISBN: 9781615043309 paperback
`
`ISBN: 9781615043316 ebook
`
`DOI: 10.4199/C00017ED1V01Y201009ISP009
`
`A Publication in the
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`CoLLoqUiUM SerieS on inTeGrATeD SYSTeMS PHYSioLoGY: FroM MoLeCULe To
`FUnCTion To DiSeASe
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`Lecture #10
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`Series Editors: Joey Granger, University of Mississippi Medical Center and D. Neil Granger, Louisiana State
`University Health Sciences Center-Shreveport
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`Series ISSN
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`print
`electronic
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`Angiogenesis
`
`ThomasH.Adair
`University ofMississippi Medical Center
`
`Jean-Pierre Montani
`University of Fribourg, Switzerland
`
`COLLOQUIUM SERIES ON INTEGRATEDSYSTEMS PHYSIOLOGY:
`FROM MOLECULETO FUNCTIONTO DISEASE #10
`
`Ne MORGAN QCLAYPOOL LIFE SCIENCES
`
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`ABSTRACT
`Angiogenesis is the growth ofblood vessels from theexisting vasculature. Thefield of angiogenesis
`has grown enormouslyin the past 30 years, with only 40 papers published in 1980 and nearly 6000
`in 2010. Whyhas there been this explosive growth in angiogenesis research? Angiogenic therapies
`provide a potential to conquer cancer, heart diseases, and more than 70oflife’s most threatening
`medical conditions. Thelives ofat least 1 billion people worldwide could be improved with angio-
`genic therapy, according to the Angiogenesis Foundation. In this little book, we provide a simple
`approach to understandtheessential elements of the angiogenic process, we critique the most pow-
`erful angiogenesis assays that are used to discover proangiogenic and antiangiogenic substances,
`and weprovide an in-depth physiological perspective on how angiogenesis is regulated in normal,
`healthy tissues of the human body. All tissues of the body require a continuous supply of oxygen to
`burn metabolic substrates that are needed for energy. Oxygen is conducted to these tissues by blood
`capillaries: more capillaries can improve tissue oxygenation and thus enhance energy production;
`fewer capillaries can lead to hypoxia and even anoxia in the tissues. This means that angiogenic
`therapies designed to control the growth and regression ofbloodcapillaries can be used to improve
`the survival of poorly perfusedtissues that are essential to the body (heart, brain, skeletal muscle,
`etc.) and to rid the body of unwantedtissues (tumors).
`
`
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`Theauthorsare grateful for the editorial assistance of Karen A. Richards and Leslie S. Adair. This
`work was supported by a grant from the National Heart, Lung, and Blood Institute (HL-51971).
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`
`
`Overview ofAngiogenesis ...........ssccsssesssecesseeesseceeeeeceeceseeeseseeeesseceeeeceeeseneeeeeseeeees 1
`LL History oo... ceeeeececceesesescseeceseeeecsesesensnenececessesessnsnsnsceenssesecseseseseusnecececeesesesanensanenecess 1
`1.2 Origin of Blood Vessels ..........s:ssssssssesesessesesesesesesceeeecscseseseseseesesceceesaeeeesesensanenecess 2
`1.3 The Angiogenic Process .........sssscsssecsssseseseesscscscssseseesescseesescecsssneessesesnsesseeseeeseees 3
`1.3.1 Types ofAngiogenesis .........c.scscccessesssescsssesessscsesesesescscecseseecseececseaneeeeecaees 3
`1.3.2 Sprouting Angiogenesis...........scscesssscssseseesseseseeceeseseseececsesesceeseecsesneceeeaees 4
`1.3.3
`Intussusceptive Angiogenesis...........cccesescsceseseseeesesessssesceceeeseseecseseeenenesees 6
`
`ATIQIOGCTICRIS ASBBYB 50...ocscscss0cscecsccsssscessovansvseecssecseessusseusonensssescssoosnsesonecusosecsseoses 9
`2.1
`In Vitro Assays.......ccccessecsssscsssesescececsesesescscecsnsesnesesessssnesesesesensnssesesansneessseessneneeees 9
`2.1.1 Endothelial Cells Are Heterogeneous..........c:c:ccesesesesessesesesesesescesnsceceeeees 9
`2.1.2 In Vitro Conditions Rarely Reflect In Vivo Environment..............0:000+ 9
`2.1.3 Endothelial Cell Proliferation Assays..........sssssssssesssssesesssesesescsnscseeees 10
`2.1.4 Endothelial Cell Migration Assays.........:cccsssssesesescscsesesesesesesssenseeeeees 11
`2.1.5 Endothelial Tube Formation Assays ........cscscsessssseeesesceseeeseseeceeesseeeees 12
`2.1.6 Rat and Mouse Aortic Ring Assay ........cccccsssessesesescscesesesesesesesseenseeeeees 14
`In Vivo Assay......cscscesesesesesesssseececeeseseseaesescscesessssecsesesesessnecacecseseassnseseneneseceses 15
`2.2.1 Corneal Angiogenesis Assay....
`veces 15
`2.2.2 Chick Chorioallantoic Membrane(CAM)‘AngiogenesisAssay...
`.. 16
`2.2.3 Matrigel Plug Assay ........s.scscsesccssesssesesesssnsnseseesenencessssesescesesseseseeeesasaeees 17
`
`2.2
`
`Regulation: Metabolic Factors ..........::scssscsssseesseeessecerseeceseseeeecsseeeseeceeeeeeeeeeneees 19
`3.1 Capillary Growth Is Proportional to Metabolic Activity.........ccccsseseeseseeeeeees 19
`3.2
`Increasing Metabolic Activity Stimulates Blood Vessel Growth... 20
`3.3. Decreasing Metabolic Activity Causes Vascular Regression....
`ssssesenes 22
`3.4 Long-Term Increases in Blood Pressure Lead to Vascular Rarefaction..
`23
`3.5 Oxygen Is a Master Signal in Growth Regulation ofthe Vascular Systenn 24
`3.5.1
`Increases in Muscular Activity Cause Decreases in
`Muscle Oxygemation..........cscccsssesssssesssescssscessesescscecsesesnscececsnsneceeseeeneneess24
`3.5.2 Oxygen Regulates Angiogenic Growth Factor Production............:000+ 24
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`x ANGIoGENESIS
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`4.
`
` VEGF-A Released From Hypoxic Tissues Is a Key Regulator
`3.5.3
`of Angiogenesis ..................................................................................... 25
`
`3.5.4 Negative Feedback Regulation of VEGF-A .......................................... 26
`3.5.5
` Oxygen Plays a Central Role in Feedback Regulation of Vascular
`
`Growth and Regression ......................................................................... 27
`3.6 Role of Adenosine in Metabolic Regulation of Vascular Growth ...................... 28
`
`Regulation: Mechanical Factors ......................................................................... 31
`4.1 Control of Blood Vessel Growth ....................................................................... 31
`4.1.1 Epithelial Sodium Channel Protein Biology ......................................... 31
`4.1.2 Epithelial Sodium Channels Can Form a Mechanosensory
`
`Complex ................................................................................................ 32
`4.1.3
` Epithelial Sodium Channels Can Mediate Mechanotransduction in
`
`Mammals ............................................................................................... 32
`4.1.4 Do Epithelial Sodium Channels Mediate Angiogenesis? ...................... 33
`4.1.5 Physical Forces Acting on the Walls of Blood Vessels ........................... 33
`4.1.6 Shear Stress Is Sensed by the Endothelium ........................................... 34
`4.1.7 Increased Blood Flow (Shear Stress) Can Stimulate Angiogenesis ....... 35
`4.1.8
` Possible Role of Endothelial Cell Shape in Regulating Blood Vessel
`
`Growth and Regression ......................................................................... 36
`4.1.9 Mechanical Factors Have an Accessory Role in Angiogenesis .............. 37
`4.2 Control of Lymphangiogenesis ......................................................................... 38
`4.2.1 Flow-Guided Lymphangiogenesis ........................................................ 39
`4.2.2 High Salt Load Stimulates Lymphangiogenesis in Skin ....................... 40
`
`Glossary .................................................................................................................... 41
`
`References ................................................................................................................. 47
`
`Author Biographies .................................................................................................... 71
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`CHAPTER 1
`
`
`
`Angiogenesis is the growth of bloodvessels from the existing vasculature. It occurs throughoutlife
`in both health and disease, beginning in utero and continuing on through old age. No metabolically
`active tissue in the body is more than a few hundred micrometers from a bloodcapillary, which is
`formedbythe process of angiogenesis. Capillaries are neededin all tissues for diffusion exchange of
`nutrients and metabolites. Changes in metabolic activity lead to proportional changes in angiogen-
`esis and, hence, proportional changes in capillarity. Oxygen plays a pivotal role in this regulation.
`Hemodynamicfactors are critical for survival ofvascular networks andforstructural adaptations of
`vesselwalls.
`
`Recognition that control of angiogenesis could have therapeutic value has stimulated great
`interest during the past 40 years. Stimulation of angiogenesis can be therapeutic in ischemic heart
`disease, peripheral arterial disease, and woundhealing. Decreasingor inhibiting angiogenesis can be
`therapeutic in cancer, ophthalmic conditions, rheumatoidarthritis, and other diseases. Capillaries
`grow andregress in healthytissues according to functional demands. Exercise stimulates angiogen-
`esis in skeletal muscle andheart. A lack of exercise leads to capillary regression. Capillaries grow
`in adipose tissue during weight gain and regress during weight loss. Clearly, angiogenesis occurs
`throughoutlife.
`
`1.1
`HISTORY
`The Scottish anatomist and surgeon John Hunterprovided thefirst recorded scientific insights into
`the field of angiogenesis. His observations suggested that proportionality between vascularity and
`metabolic requirements occurs in both health and disease. This belief is summarized in his Treatise
`published in 1794 [1] as follows: “In short, whenever Nature has considerable operations going on,
`andthose are rapid, then wefind the vascular system in a proportionable degree enlarged.” Although
`the term angiogenesis does not appear in his writings [1,2], Hunter was the first to recognize that
`overall regulation of angiogenesis follows a basic law of nature founded byAristotle [3], which in es-
`sence is “form follows function.” The modern history of angiogenesis began with the work ofJudah
`Folkman, who hypothesized(and published in 1971) that tumor growth is angiogenesis-dependent
`[4]. Recognition that control of angiogenesis could lead to cancer therapies stimulated intensive
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`2 ANGIoGENESIS
`
`research in the field, e.g., only two manuscripts dealing with angiogenesis were published in 1970 and
`over 5200 articles were published in 2009. For detailed histories of angiogenesis, see Refs. [5–11].
`
`1.2 oRIGIN oF BLooD VESSELS
`The cardiovascular system is the first organ system to develop in the embryo [12]. The luminal
`surface of the circulatory system in contact with blood is a single layer of
`: these
`are derived from
` (Figure 1.1).
` differentiate from
`
`and give rise to
` and
`. Angioblasts are a cell type with potency
`to differentiate into endothelial cells but have not yet acquired all characteristic markers of endo-
`thelial cells.
` (Figure 1.2) is the de novo formation of blood vessels from
`
`[12–14]. It occurs in the extraembryonic and intraembryonic tissues of embryos [12,14]. Vasculo-
`genesis is a dynamic process that involves cell–cell and cell–
` (ECM) interactions
`directed spatially and temporally by
` and
` [14–17]. This process includes
`differentiation of mesodermal stem cells into angioblasts, growth factor directed migration of angio-
`blasts to form
` where angioblasts give rise to
` [12–14].
`.
`,
`Other types of vascular growth include
`, and
`The term
` means the formation of any blood vessel in the adult regardless of its
`size or type.
`
`FIGURE 1.1: Origin of endothelial cells and hematopoietic cells [14]. Mesodermal stem cells are the
`source of hematopoietic stem cells and angioblasts in the developing embryo. The hemangioblast is a
`precursor to both angioblasts and hematopoietic stem cells. Angioblasts differentiate into endothelial
`cells. Hematopoietic cells can differentiate into all cell types found in circulating blood
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`endothelial cells
`
`mesoderm
`
`Hemangioblasts
`
`mesodermal stem cells
`
`hematopoietic stem cells
`
`angioblasts
`
`Vasculogenesis
`
`angioblasts
`
`extracellular matrix
`
`growth factors
`
`morphogens
`
`blood islands
`
`endothelial cells
`
`neovascularization
`
`arteriogenesis
`
`venogenesis
`
`lymphangiogenesis
`
`arteriogenesis
`
`arteriogenesis
`
`venogenesis
`
`venogenesis
`
`lymphangiogenesis
`
`lymphangiogenesis
`
`

`

`oVERVIEw oF ANGIoGENESIS 3
`
`FIGURE 1.2: Vasculogenesis in the vertebrate embryo. (a) Angioblasts derived from lateral mesoderm
`are committed to become arteries (red) or veins (blue). The cardinal veins assemble from precursor cells
`(blue) that remain in a lateral position. (b) Artery precursor cells migrate toward a vascular endothelial
`growth factor type A (
`) stimulus secreted from cells in the midline. (c) The migrating arterial
`angioblasts align into cords forming a plexus. (d) Arterial angioblasts coalesce forming the dorsal aorta.
`(e) Intersomite vessels are assembled from three types of endothelial cells with different morphologies
`indicated as blue, purple, and green. Used with permission from Nature Publishing Group: Hogan
`(2002) [18].
`
`THE ANGIoGENIC PRoCESS
`1.3
`1.3.1 Types of Angiogenesis
`Sprouting angiogenesis and intussusceptive angiogenesis both occur in utero and in adults. Sprout-
`ing angiogenesis is better understood having been discovered nearly 200 years ago: intussusceptive
`angiogenesis was discovered by Burri [19,20] about two decades ago. Figure 1.3 shows the basic
`morphological events for both types of angiogenesis. As implied by its name, sprouting angiogenesis
`is characterized by sprouts composed of endothelial cells, which usually grow toward an angiogenic
`stimulus such as VEGF-A. Sprouting angiogenesis can therefore add blood vessels to portions of
`tissues previously devoid of blood vessels. On the other hand, intussusceptive angiogenesis involves
`formation of blood vessels by a splitting process in which elements of interstitial tissues invade exist-
`ing vessels, forming transvascular tissue pillars that expand. Both types of angiogenesis are thought
`to occur in virtually all tissues and organs.
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`4 ANGIoGENESIS
`
`FIGURE 1.3: Basic types of primary vascular growth. Redrawn after Carmeliet and Collen (2000) [21].
`
`1.3.2 Sprouting Angiogenesis
`The basic steps of sprouting angiogenesis include enzymatic degradation of capillary basement
`membrane, endothelial cell (EC) proliferation, directed migration of ECs, tubulogenesis (EC tube
`formation), vessel fusion, vessel pruning, and pericyte stabilization. Sprouting angiogenesis is ini-
`tiated in poorly perfused tissues when oxygen sensing mechanisms detect a level of
` that
`demands the formation of new blood vessels to satisfy the metabolic requirements of
`
` (Figure 1.4). Most types of parenchymal cells (myocytes, hepatocytes, neurons, astrocytes, etc.)
`respond to a hypoxic environment by secreting a key proangiogenic growth factor called vascular
`endothelial growth factor (VEGF-A). There does not appear to be redundant growth factor mecha-
`nisms that can replace the role of VEGF-A in hypoxia-induced angiogenesis.
`An endothelial
` guides the developing capillary sprout through the ECM toward an
`angiogenic stimulus such as VEGF-A [22–25]. Long, thin cellular processes on tip cells called
` secrete large amounts of proteolytic enzymes, which digest a pathway through the ECM
`for the developing sprout [26,27]. The filopodia of tip cells are heavily endowed with VEGF-A
`receptors (
`), allowing them to “sense” differences in VEGF-A concentrations and causing
`them to align with the VEGF-A gradient (Figure 1.5). When a sufficient number of filopodia on
`a given tip cell have anchored to the substratum, contraction of actin filaments within the filopodia
`literally pull the tip cell along toward the VEGF-A stimulus. Meanwhile, endothelial
`
`proliferate as they follow behind a tip cell causing the capillary sprout to elongate. Vacuoles develop
`and coalesce, forming a lumen within a series of stalk cells. These stalk cells become the trunk of the
`newly formed capillary. When the tip cells of two or more capillary sprouts converge at the source of
`VEGF-A secretion, the tip cells fuse together creating a continuous lumen through which oxygen-
`ated blood can flow. When the local tissues receive adequate amounts of oxygen, VEGF-A levels
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`hypoxia
`
`parenchymal
`
`cells
`
`tip cell
`
`filopodia
`
`VEGFR2
`
`stalk cells
`
`

`

`oVERVIEw oF ANGIoGENESIS 5
`
`FIGURE 1.4: VEGF-A directed capillary growth to poorly perfused tissues. (A) Endothelial cells ex-
`posed to the highest VEGF-A concentration become tip cells (green). Hypoxic tissue is indicated by
`the circular blue fade. (B) The tip cells lead the developing sprout by extending numerous filopodia.
`(C) The developing spout elongates by proliferation of endothelial stalk cells (purple) that trail behind
`the tip cell. (D) The tip cells from two developing sprouts fuse and create a lumen. (E) Blood flowing
`through the new capillary oxygenates the tissues, thus reducing the secretion of VEGF-A. (F) The newly
`developed capillary is stabilized by pericyte recruitment (red), deposition of ECM (gray), shear stress and
`other mechanical forces associated with blood flow and blood pressure. Redrawn after Carmeliet et al.
`(2009) [24].
`
`
`
`return to near normal. Maturation and stabilization of the capillary requires recruitment of
`and deposition of ECM along with
` and other mechanical signals [28].
`Delta-Notch signaling is a key component of sprout formation (Figure 1.5). It is a cell–cell sig-
`naling system in which the ligand,
` (Dll4) mates with its
` on neighboring
`cells. Both the receptor and ligand is cell bound and thus act only through cell–cell contact. VEGF-A
`induces Dll4 production by tip cells, which leads to activation of notch receptors in stalk cells.
`Notch receptor activation suppresses VEGFR2 production in stalk cells, which dampens migratory
`behavior compared with that of tip cells. Hence, endothelial cells exposed to the highest VEGF-A
`concentration are most likely to become tip cells [24,25,30]. Although tip cells are exposed to the
`highest VEGF-A concentration, their rate of proliferation is far less compared with that of stalk
`cells.
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`pericytes
`
`shear stress
`
`Delta-like-4
`
`notch receptor
`
`

`

`6 ANGIoGENESIS
`
`FIGURE 1.5: Microanatomy of a capillary sprout and tip cell selection. (A) An interstitial gradient for
`VEGF-A and an endothelial cell gradient for VEGFR2 are shown. Tip cell migration is thought to de-
`pend upon the VEGF-A gradient and stalk cell proliferation is thought to be regulated by the VEGF-A
`concentration. Redrawn after Carmeliet and Tessier-Lavigne (2005) [29]. (B) Delta-Notch signaling is
`critical for tip cell selection. Activation of notch receptors on stalk cells induces proteolytic cleavage and
`release of the intracellular domain, which enters the nucleus and decreases gene expression of VEGFR2.
`National Institutes of Health, public domain image.
`
`Not all aspects of the Delta-Notch signaling pathway are fully understood, but it is clear
`that production of a normal vasculature is heavily dependent upon the concentration of VEGF-A
`in the tissues. A 50% reduction of VEGF-A expression is lethal embryonically because of vascular
`defects [31,32], and excess VEGF-A in tumors induces overproduction of tip cells leading to a dis-
`organized vasculature [33]. This critical dependence on physiological concentrations of VEGF-A
`for construction of viable blood vessels might help explain why attempts to induce angiogenesis
`in poorly perfused tissues with VEGF-A administration and gene therapy have not been highly
`successful.
`
`1.3.3 Intussusceptive Angiogenesis
`Intussusceptive angiogenesis is also called splitting angiogenesis because the vessel wall extends into
`the lumen causing a single vessel to split in two. This type of angiogenesis is thought to be fast and
`efficient compared with sprouting angiogenesis because, initially, it only requires reorganization of
`existing endothelial cells and does not rely on immediate endothelial proliferation or migration.
`Intussusceptive angiogenesis occurs throughout life but plays a prominent role in vascular develop-
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`oVERVIEw oF ANGIoGENESIS 7
`
`ment in embryos where growth is fast and resources are limited [34–36]. However, intussusception
`mainly causes new capillaries to develop where capillaries already exist.
`Evidence for the occurrence of intussusceptive angiogenesis is based upon the presence of
`transcapillary tissue pillars (Figure 1.6). Identification of tissue pillars requires scanning electron
`micrographs of vascular casts or three-dimensional reconstruction of serial micrographs. This type
`of angiogenesis was discovered in postnatal lungs of rats and humans [19,20], but it also occurs in
`many other tissues and organs, especially in capillary networks that abut an epithelial surface, e.g.,
`choroid of the eye, vascular baskets around glands, intestinal mucosa, kidney, ovary, and uterus
`[37,38]. It also occurs in skeletal muscle, heart, and brain. In addition to forming new capillary
`structures, intussusceptive growth plays a major role in the formation of artery and vein bifurcations
`as well as pruning of larger microvessels.
`The control of intussusceptive angiogenesis is poorly understood compared with sprouting
`angiogenesis. This difference is only partly due to its recent discovery in 1986 [20]. A rate-limiting
`step in intussusceptive growth research can be pinned to the laborious methods required to
`prove its presence, which, again, involve determining the frequency of tissue pillars from scan-
`ning electron micrographs of vascular casts. However, it is known that intussusceptive angiogenesis
`can be stimulated in the chick
` (CAM) with application of VEGF-A
`(Figure 1.7), and there is little doubt that many growth factors and signaling systems are involved
`[34,37]. Mechanical stresses related to increases in blood flow can initiate intussusceptive growth in
`some high flow regions of the circulation, as discussed in Chapter 4 [34,35].
`
`FIGURE 1.6: Scanning electron micrographs of Mercox casts. (a) Fetal chicken lung microvasculature.
`(b) Rat lung microvasculature at postnatal day 44. The small holes indicated by arrows have diameters
`of about 2 µM. The holes correspond to tissue pillars that extend across the capillary lumina. Scale bars:
`(a) 12 and (b) 20 µM. Used with permission from Wiley-Blackwell: Djonov, Kurz, and Burri (2003) [35].
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`chorioallantoic membrane
`
`

`

`8 ANGIoGENESIS
`
`FIGURE 1.7: Intussusceptive angiogenesis in three dimensions (a–d) and two dimensions (a'–d'). (a,b,
`a',b') The process begins with protrusion of opposing endothelial cells into the capillary lumen. (c,c') An
`interendothelial contact is established and endothelial junctions are reorganized. (d,d') The endothelial
`(EC) bilayer and basement membranes (BM) are perforated centrally allowing growth factors to enter.
`Fibroblasts (Fb) and pericytes (Pr) migrate into the site of perforation where they produce collagen
`fibrils (Co) and other components of ECM forming a tissue pillar. Used with permission from Wiley-
`Blackwell: Djonov, Kurz, and Burri (2003) [35].
`
`• • • •
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`CHAPTER 2
`
`
`
`The most significant advancements in angiogenesis in the past 25 years have been the discovery
`of proangiogenic and antiangiogenic molecules. The development of angiogenesis assays has been
`essential to the discovery of these molecules. In vitro assays have been expeditious and quantitative
`but should be viewedasfirst approximationsthat need to be confirmedin vivo. In vivo tests are more
`time-consuming anddifficult to quantitate, but because of the complex interactions between mul-
`tiple cell types necessary to form functional bloodvessels,all in vitro findings need to be confirmed
`in the intact animal.
`
`INVITRO ASSAYS
`2.1.
`2.1.1 Endothelial Cells Are Heterogeneous
`Most endothelial cell assays utilize human umbilical vein endothelial cells (HUVECs) or bovine
`aortic endothelial cells (BAECs) not becausethesecells are good representatives ofvascular endo-
`thelial cells in vivo, but because they arerelatively easy to harvest from large blood vessels. Endo-
`thelial cells are heterogeneous [39-43]: there are differences among endothelial cells from large and
`small blood vessels, arteries, and veins, species differences, organ differences, differences between
`host and tumor, andeven differences within a given organ. Also, endothelial cells used in laboratories
`are virtually alwaysin a proliferative state rather than the normal quiescentstate of the established
`vasculature in the intact animal. Even most “primary cultures” of endothelial cells require extensive
`proliferation to obtain enoughcells for an experiment with the assumption that they retain their
`normal in vivo physiological characteristics. This assumption is often incorrect. It is well-known
`that cells in vitro can both gain andlose attributes compared with parentcells in intact animals.
`
`2.1.2 In Vitro Conditions Rarely Reflect In Vivo Environment
`Anotherissue is the environmental conditions in which endothelial cells are cultured. Endothelial
`cells in vivo are exposed to shear stress and other hemodynamic forces that activate multiple sig-
`naling pathways. Endothelial cells in vitro are usually cultured in room air (21% oxygen), which
`is hyperoxic compared with the in vivo oxygen tension, especially in the microcirculation. Two-
`and three-dimensional scaffolds using Matrigel, collagen,or fibrin are used to simulate the normal
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`10
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` ANGIoGENESIS
`
`FIGURE 2.1: (a–c) Human umbilical vein endothelial cells (HUVEC) and (d–f ) human dermal micro-
`vascular endothelial cells (HuDMEC). (a, d) Phase contrast microscopy. (b, e) CD31 or (c, f ) von Wille-
`brand factor staining: nuclei are counterstained with DAPI. Photographs courtesy of Promocell GmbH,
`Heidelberg, Germany. Used with permission from Wiley-Blackwell: Staton et al. (2009) [42].
`
`extracellular matrix, but it is clear that complex interactions between endothelial cells and their in
`vivo physical environment cannot be fully simulated in culture. Also, in vivo interactions between
`endothelial cells and other cell types (smooth muscle cells, pericytes, fibroblasts, macrophages, etc.)
`are difficult to simulate in vitro. For these reasons, in vitro angiogenesis assays should be viewed as
`a starting point rather than an endpoint for discovery, depending on the purpose of the experiment
`(Figure 2.1).
`
`2.1.3 Endothelial Cell Proliferation Assays
`Proliferation of endothelial cells is needed for developing capillaries in the intact animal. The ac-
`tions of proangiogenic and antiangiogenic molecules on proliferation can be assessed by direct cell
`counts, DNA synthesis, or metabolic activity [39–42,44]. For testing potential proangiogenic mol-
`ecules, it is often necessary to reduce the rate of proliferation by decreasing serum levels in culture
`media, and it is usually more effective to test antiangiogenic molecules on cells that have a substan-
`tial rate of proliferation.
`The rate of cell proliferation can be determined by counting cells at 24-h intervals after seed-
`ing multiple cultures with a defined number of cells (Figure 2.2). Cells can be counted using a hemo-
`cytometer and light microscope or an electronic Coulter counter or similar device. Cell proliferation
`is often determined using a colorimetric method (MTT assay) in which mitochondrial enzymes
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`ANGIoGENESIS ASSAyS 11
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`FIGURE 2.2: Typical growth curve for HUVECs in culture media containing 10% fetal bovine se-
`rum (FBS). Media were changed daily. Cells were counted using a Coulter counter. Redrawn after Lee
`(2006) [44].
`
`reduce MTT to formazan dyes in proportion to cell number. MTT (3-(4,5-dimethylthiazol-2-yl)-
`2,5-diphenyltetrazolium bromide) is a yellow tetrazole that is reduced to purple formazan in living
`cells: absorbance is read by a spectrophotometer. DNA synthesis is often used to reflect cell prolif-
`eration. With the commonly used [3H]thymidine incorporation method, the amount of radioactiv-
`ity in cells is proportional to the amount of newly synthesized DNA. A similar but nonradioactive
`method utilizes bromodeoxyuridine (BrdU), which competes with thymidine for incorporation into
`DNA. Despite their popularity, these latter methods are not fully reliable. Changes in metabolic
`rates of individual cells and/or compounds that affect mitochondrial enzyme activity can cause gross
`miscalculations of cell proliferation with the MMT assay, and several investiga