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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 AcademicPressis animprintofElsevier 125LondonWall,LondonEC2Y 5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101, UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA 02139,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom Copyright©2022 ElsevierInc.Allrights reserved. 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ISBN:978-0-12-824371-8 Forinformation onallAcademicPress publicationsvisitourwebsiteat https://www.elsevier.com/books-and-journals Publisher:StacyMasucci Acquisitions Editor:Rafael E.Teixeira EditorialProjectManager: SaraPianavilla ProductionProjectManager: KiruthikaGovindaraju CoverDesigner: MilesHitchen TypesetbyTNQTechnologies 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, between 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 vertebrates, 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 definition 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 structures 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 developed 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 circulatory 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 amoebocytes 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