The Vascular Endothelium: A Holistic Approach for Oncology

136 Pages·2022·7.657 MB·English

by  Domenico Ribatti, Francesco Pezzella

The Vascular Endothelium
A Holistic Approach for Oncology
Domenico Ribatti
Professor of Human Anatomy
Department of Basic Biomedical Sciences
Neurosciences and Sensory Organs
Section of Human Anatomy and Histology
University of Bari Medical School
Bari, BA, Italy
Francesco Pezzella
Professor of Tumour Pathology
Nuffield Division of Clinical Laboratory Science
Radcliffe Department of Medicine
University of Oxford
United Kingdom
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1
Appearance and evolution of the
endothelial cell
1.1 Appearance of a circulatory system
Once upon a time there was no circulatory system but the need for it arose with the
development of multicellular organisms. Living organisms are divided, according to
current Taxonomy, into three groups called Domains: Bacteria, Archea end Eukarya
(Woese et al., 1990). The Domain Eukarya is divided into seven Supergroups. The
Supergroup Land Plants account for the Kingdom Plants while the supergroup
Opisthokonta (meaning with “posterium flagellum”) contains two other Kingdoms:
Fungi and Animalia (Metazoa) (Brooker et al., 2014). The supergroup Opisthokonta is
characterizedbythefactthat,whenpresentinthecomponentsofthisgroup,cellswitha
flagellum are able to use it to propel themselves. While unicellular organisms directly
exchange nutrients, metabolite and catabolite with the medium they inhabit, the
development of larger and/or thicker than, approximately, 1mm organisms has been
conditional to establish an adequate system for distribution and disposal of nutrients
plus any other molecules linked to their vital functions (Munoz-Chapuli & Perez-
Pomares, 2010). Such systems are found both in the Plant and Animal kingdoms
although they present very different characteristics.
In the animal kingdom (Fig. 1.1), the vascular system first appeared around 600
millionyearsago.Whilesmallerthanapproximately1or2millimeters,Metazoancould
rely effectively on diffusion, larger multicellular organisms needed “cavities” inside the
organismallowingfluidstocirculateandtransportmoleculesacrossthebodyaccording
to requirement (Monahan-Earley et al., 2013). The endothelial cells evolved later, be-
tween 540 and 510 million year ago, when Vertebrates, the largest subphylum of the
Cordate phylum, are believed to have separated from the other two Cordate subphilia,
the Urochordata and Cephalochordata (Bikfalvi, 2016). At the same time of the
appearance of the endothelium, endothelial heterogeneity also developed (Monahan-
Earley et al., 2013) as supported by the fact that in the Hagfish, the oldest living verte-
brates, the phenotype of the endothelial cells differs through the body from organ to
organ (Cheruvu et al., 2007; Shigei et al., 2001).
Presently, in the literature, an endothelial cell is designated as the one covering the
luminal space of the vessels, forming a continuous layer (except in some specialized
vessel like liver sinusoids) having a basal/luminal polarity and these cells are kept
adherent to each other, and to the basement membrane, by specialized junctional
TheVascularEndothelium.https://doi.org/10.1016/B978-0-12-824371-8.00003-7 1
Copyright©2022ElsevierInc.Allrightsreserved.
2 The Vascular Endothelium
FIGURE 1.1 All the diploblasts and the simpler triploblasts do not have coeloma which appears in large
invertebrates(e.g.,theEarthworms).Themorecomplexinvertebratesevolved,thankstothedevelopmentofthe
Hemel,apropervascularsystemwithheart/heratlikestructures.Theendotheliumisinsteadacharacteristicofthe
vertebrates:onlythem,andallofthe,haveendothelialcells.
complexes (Munoz-Chapuli et al., 2005). It is a striking fact that, when using this defi-
nition of endothelium, all the papers published in literature report, and conclude, that
vertebrates have proper endothelial cells while the invertebrates do not. It is therefore
the current accepted wisdom that the presence of cells called “endothelial,” according
totheabovedefinition,dividesinvertebratesfromvertebrates(Cheruvuetal.,2007),that
is, the endothelium and the backbone appear to have evolved together.
InthesimplestorganismsliketheDipoblasts(Metazoathoseembryoshaveonlytwo
layers, the ectoderm and the endoderm), no coelomic cavity or other circulatory struc-
tures are seen. Some of the smallest Triploblasts (animals with three embryonal layers:
ectoderm,mesodermandendoderm)alsodonothavecoelomaticcavity.Thischamber,
the simplest cavitated system to circulate fluids inside a body, starts to appear as the
Triploblasts increased in size (Hartenstein & Mandal, 2006). The earliest vessels devel-
oped around the gut to collect and transport nutrients, rather than oxygen, with the
gases exchange happening through the skin. Later on during evolution, vessels also
startedtotransport andexchangegases.Evolutionoftheallcardiovascularapparatusis
a vast topic (Burggren & Reiber, 2007); therefore, in this chapter, we will focus on the
evolutionary development of the endothelial cell. As there are no fossils remains of
endothelium, its evolutionary history rely mostly on molecular phylogeny, that is, the
comparison of genetic material between species to understand their evolutionary rela-
tionship,andonontogenesis,thestudyofthedevelopmentofexistingorganisms(Aird&
Laubichler, 2007; McVey, 2007).
Chapter 1 (cid:1) Appearance and evolution of the endothelial cell 3
1.2 Coeloma, the basic circulatory system
Asmentionedabove,earlymulticellularorganismslikeflatwormsstillrelyondiffusion.
Astheydonothaveevenacoelomaticcavity,theyarealsoclassifiedasAcoelomates:all
theorgansareembeddedinsideamesenchymaltissueandliquid-filled,ofanytype,are
absent (Conn, 1993) although some occasional small poorly defined liquid filled spaces
can be recognized (Pseudocoelomate) (Monahan-Earley et al., 2013) (Figs. 1.2 and 1.3).
To grow bigger than an Acoelomate, multicellular organisms had to develop a cir-
culatory system, as simple diffusion would not suffice. This happened sometime before
600millionofyearsago(Bikfalvi,2016).Aroundthistime,largerorganismswithafluid-
filledbodycavity,the“coelom,”evolved,forexample,theearthworms.Thewallsofthis
spaceareformedbytissuefromthemesodermandarelinedupwithcellswhichprevent
theleakingofthefluidfromthecavityintothesurroundingmesenchyma.Thesecellsare
known as mesothelial, that is, epithelial cells of mesodermal origin, to distinguish them
from epithelial cells of either ectodermal or endodermal origin (Bikfalvi, 2016; Holland,
2011).Manyofthesecellshaveciliathosemovementscontributetothecirculationofthe
fluid.Insomemetazoan,thesecoelom-liningcellscanhavemyo-epithelialfeatures,that
FIGURE 1.2 Basic anatomy of the circulatory systems. (A) Acoelomata diploblasts metazoan has only two
embryonallayers:ectodermandendoderm.Thetwoepitheliallayersformadigestivetubeandtheexternal
epithelium.Thereisnomesenchymaandnocoelomaticcavity.(B)Acoelomatatriploblastshavethreeembryonal
layers,ectoderm,endoderm,andmesoderm.(C)Pseudocoelomatawithsomespacesfilledwithfluidbutno
mesotheliallining.
4 The Vascular Endothelium
FIGURE1.3 Basic anatomy ofthecirculatory systems.(A)Coelomatesaremetazoan with acolema: aspace lined
upbymestotheliumfilledupbyfluidusuallycontainingsomehematiccells.Themesotheliumcanhave
myoepithelialfeaturesormusclelikecells,whicharepresentinthenearbymesenchymaprovidingcontractor
movements.(B)Inlargeinvertebrates,theHemelcirculatorysystemdevelops.Thesechannelsarelinedby
mesodermalextracellularmatrix.Someinvertebratescanhavesomecellsaliningtractsofvesselsbuttheseare
Amoebocytesanddonothaveallthecharacteristicsoftheendothelialcells.(C)Vertebrates.Thecirculatory
systemsofallvertebratesareinsteadlinedupbyacontinuouslayerofendothelialcells.Coelomaticcavitiesare
stillpresentbutdonothaveanyroleincirculation.
is, they can also contract increasing the movement of the fluid. In other organisms
instead, there are proper muscle cells surrounding the coelom and sustaining the fluid
recirculation, with their contractions (Munoz-Chapuli & Perez-Pomares, 2010). Cavity
lined by either mesoderm, in some areas, or endoderm, in other locations, are called
pseudocoelom (Brooker et al., 2014), not to be confused with the Pseudocoelomate
spaces described in the Acoelomate as described above.
The coelom has several functions, alongside acting as a primordial circulatory
system, including physical protection of internal organs and offering a space for these
organs to grow and move in. Nutrients and gasses diffuse from the skin to the fluid
inside the cavity, and the fluid is then kept in motion spreading equally the solutes
throughthebody.Atthesametime,anysubstancetobeexcretedisreleasedinsidethe
fluid and subsequently expelled, by diffusion, through the skin. The Coelom is still
persistent in larger animals and, in mammals, has developed into the peritoneal,
pleural, and pericardiac cavities. These spaces have a fundamental role in
allowing organs like the heart or the intestine to move freely while performing their
functions.
Chapter 1 (cid:1) Appearance and evolution of the endothelial cell 5
1.3 The hemel, vascular channel without endothelium
AlongsidetheCoelom,achannel-basedcirculatorysystemstartedalsotoevolve,theso-
called Hemal System: this is defined by three components: a system of vessels, that is,
hollow tubes, a special fluid filling it (blood, hemo-lymph, or lymph) and, finally, the
presence of one or more of a modified vascular segment, rich in muscle cells, able to
pumpthefluid:theheartanditsprecursors(Brookeretal.,2014)(Figs.1.2and1.3).The
cellular component of the Hemel system, mainly the myoepithelial and the circulating
cells, is regarded as originating from the coelom epithelium, both in phylogenesis and
ontogenesis (Munoz-Chapuli & Perez-Pomares, 2010).
Why was the coelom not enough and, therefore, why did the hemel evolved? One
hypothesis is that, when segmented animals started to appear, the formation of vessels
was necessary to allow the transport of fluid from the coelom of one segment to that of
another one (Monahan-Earley et al., 2013). The presence of an early vascular system
does not rule out completely diffusion or the presence of a working coelom (Ruppert &
Carle,1983):forexample,thehemelsystemininsectsdoesnotdeliveryoxygen,andthis
is done by the tracheal system. The combined hemel-coelom system is found in some
invertebrate: in these animals there are heart structures, from which vessels originate,
these vessels conduct the fluid to an open cavity, the hemocoel. Here, the fluid, called
hemolympha, is subject to exchange of nutrients, catabolites and gas than the fluid
return tothe heart. Two arethe anatomicalstructuresdeliveringthe hemolymphafrom
hemocoelto the heartthat canbefound.Insome organisms,the hemocel is connected
to the heart by some downstream channels while in others the hemocel opens directly
inside the heart. In more complex animals instead, the vessels and the coelom cavities
are separated and an anatomical communication between the two is no longer present
(Brooker et al., 2014).
In most invertebrate, the vascular luminal side of their channels is not covered by
cells but it is lined by mesodermal matrix (Munoz-Chapuli et al., 2005; Pascual-Anaya
et al., 2013). Some invertebrates have an incomplete lining of endothelial-like cells
(Ruppert & Travis, 1983) which are of mesothelial/epithelial or myoepithelial in origin
(Munoz-Chapuli et al., 2005). However, crucially, these cells do not have junctional
complexesattachingthemtooneanothernoradefinedpolarity.Thesimplestorganisms
withafullylinedvascularsystembelongtothephylumNemerteans(orRibbonWorms).
These cells covering the vascular wall are myo-epithelial with cilia on the luminal side
and it has been proposed that are close to those liningthe coelomatic cavity, leading to
the hypothesis that these vessels could evolve celomatic spaces (Burggren & Reiber,
2007). These myoepithelial cells can also be present in the vascular channel (hemal
spaces) of several other invertebrates and they are likely to be the ancestors of the
pericytespresentinvertebrates(Munoz-Chapulietal.,2005).Invertebrates,adamageto
endothelium isone ofthe causes ofblood clotting butthe invertebrateshave arange of
different clotting system which allows to their blood not to clot despite the lack of
endothelium. This is possible as the first activation steps are different: instead of fibrin,
6 The Vascular Endothelium
thrombin, and von Willebrand factor, other molecules are involved. These coagulation
pathways change in different species of invertebrates and, for example, in insects are
based on fondue, phenoloxidase, and transglutaminase while in crabs are the
lipoopolisacaride family and the protein C (Bikfalvi, 2016).
The cells populating the blood of the invertebrates belong to a large variety of types,
with many different functions. This heterogeneous group of cells has been divided into
four main types: the prohemocytes (progenitors), hyaline hemocytes (plasmatocytes or
monocytes), granular hemocytes (granulocytes), and eleocytes (containing fat droplets)
(Hartenstein, 2006). A special type of cells found circulating in the channels of some
invertebrates, and able to adhere to the channel walls, are those which belong to the
hyalinehemocytestypeandarecalledamebocyte(Monahan-Earleyetal.,2013;Munoz-
Chapuli et al., 2005). It is not a well-defined group of cells as, for example, the name
“amebocytes” has been used to describe a different subset of cells present in the mes-
enchyma of the acoelomic metazoan (Hartenstein & Mandal, 2006). The same authors
also warn that the hemocytes are high variable among different invertebrates and that,
on the other hand, the same cells can receive different names by different authors give
the extreme complexity of this topic and the lack of a complete standardized
nomenclature.
Theamoebocytes(speltalsoamebocytes),accordingtothemostcommondefinition,
that is, cells belonging to the hyaline hemocytes, are found in the circulating fluids of
many different invertebrate, but also in other locations like epithelia or mesoglea. They
have variable morphology and usually contain a high number of granules (Gold &
Jacobs, 2013). Most of what we know about amoebocytes comes from ultrastructural
studies and not much is known about their genetic and phylogenesis or their role in
development of other cell types. Their more obvious function seems to be in inflam-
mationandimmunity:inseveralinvertebrateorganisms,thesecellshavebeenfound,on
histological examination, to display a phagocytic activity in response to tissue damage,
introduction of foreign material, or infective agents (Gold & Jacobs, 2013). As amoebo-
cytes show many variations, not all are phagocytic, and they can have completely
different functions, like production of melanin, or association with oocytes and sper-
matogenesis. It is currently assumed that amoebocytes themselves can have different
degrees of differentiation, and what we call amoebocytes is regarded as a rather big,
heterogeneousgroupofcells(Gold&Jacobs,2013).Fromnowonwewillthereforerefer,
when talking about amoebocytes only to the one related to the circulatory system.
Circulating amoebocytes have been described in earthworms where they can also
adhere to the acellular luminal surface of its vessels. They can remain rounded or
become flat, are granulated, and are able to incorporate tracers suggesting, again, the
presence of phagocytic activity (Gold & Jacobs, 2013; Monahan-Earley et al., 2013). In
some invertebrates, like Holothurians and Cephalopods, the amoebocytes lining the
vascularwallsaresonumeroustoproducean“endothelium-likeappearance”;however,
they lack intercellular junctions and polarity (Munoz-Chapuli et al., 2005). These
amoebocytes are considered to be the invertebrate equivalent of the macrophages
Chapter 1 (cid:1) Appearance and evolution of the endothelial cell 7
(Ottaviani, 2011). In the Ark clam (Anadara Kagoshimensis), the hemocytes, defined as
cells found in the circulating fluid, are of three morphological types: amoebocytes,
erythrocytes, and intermediate (Kladchenko et al., 2020). The amoebocytes are baso-
philic while the intermediate are large, esosinophilic cells. The erythrocytes have
dimension similar to that of the intermediate cells and, by definition, contain hemo-
globin. All these hemocytes appear to lack G2 phase and are quiescent. They produce
Reactive Oxygen Species (ROS). Further investigation by flow cytometry demonstrated
that the amoebocytes are one heterogeneous population for dimension and number of
granules (Kladchenko et al., 2020).
But what precursor cell the amoebocytes develop from? With ultrastructural studies
Jeongproposed that therearecellcolonies, hecalled “Amebocyteforming organ,” from
which,inBiomphalariaGlabrata,hemocytes/amoebocytesarereleasedandheproposed
that these colonies are bone marrow equivalent (Jeong et al., 1983). Other authors
disagreeandreportthathemocytesappeartooriginatefromnichesofthemesenchyma
well distinct from the anatomical areas generating amoebocytes (Souza & Andrade,
2006). In the horseshoe crab (Lymulus Polyphemus), instead there is only one type of
hematocytes and this is classified as Amoebocytes (Coursey et al., 2003).
Genetic studies have determined a basic gene network shared between the hemal
system of the invertebrate and the endothelium-lined system of the vertebrate sup-
porting a common origin (Aird & Laubichler, 2007; Pascual-Anaya et al., 2013).
1.4 The oldest endothelium
The living vertebrates (Phylum Cordata, Sub-phylum Vertebrate) divide into the intra-
phylum of the Agnatha (without jaw) and that of the Gnathostomes (with the jaw). The
formeristhensplitintotheMixinidaeFamily,theHagfishgenus,andtheLampreygenus,
OrderofthePetromyzontiformes.Allthevertebrateshaveendothelium,aspreviouslydis-
cussed,liningtheirvessels(Figs.1.2and1.3)suggestingthatitfirstappearedintheirim-
mediateancestor,theLastVertebrateCommonAncestor,supposedtohavelivedbetween
510 and 550 millions of years ago. The Gnathostomes account for most of the vertebrate
specieswhiletheothertwogroupsaresmaller,astheHagfisharemadeupby67speciesand
the Lampreys by approximately 40 (Janvier, 2010). The Hagfish genus is regarded as the
closest to the Last Vertebrate Common Ancestor (Cheruvu et al., 2007; Monahan-Earley
etal.,2013).HagfishhaveashapesimilartothatoftheEels:theydohaveacraniumand
thereisnopropercompletevertebralcolumn,onlysomeill-formedvertebraearepresent.It
hasbeenargued,sincethe19thcentury,whetherHagfishandLampreywereearlytypeof
vertebrates evolving into Gnathostomes or whether they represented offshoots with a
degenerationofspinalstructure,suggestingthattheLastVertebrateCommonAncestorwas
actuallymorecomplexthatHagfishandLampreyare(Janvier,2010;Heimbergetal.,2010).
The ventral aorta and the other large vessels of the Hagfish have three layers: tunica
adventitia, media, and endothelium. The aortic endothelial cells are flattened or