Tumor Angiogenesis: A Key Target for Cancer Therapy

304 Pages·6.337 MB·English

by  Dieter Marmé (eds.)

Mechanisms of Tumor Angiogenesis
Iratxe Zuazo-Gaztelu and Oriol Casanovas
Abstract activator of the angiogenic switch, its regula-
Tumor angiogenesis, the process by which tion and a detailed description of normal and
bloodvesselspenetrateandgrowinthetumor aberrant tumor vessels are also provided. The
microenvironment,isessentialforoxygenand understanding of the foundations of these
nutrient supply and hence constitutes a key mechanisms is crucial for an effective thera-
playerforthesurvivalofsolidneoplasms.Dif- peutictargetingoftheangiogenicprocess.
ferent mechanisms of angiogenesis are devel-
oped during tumor progression such as
Keywords
vasculogenesis,sproutingangiogenesis,intus- Physiologicalangiogenesis(cid:129)Tumorangiogen-
susception, and vasculogenic mimicry. The esis (cid:129) Sprouting angiogenesis (cid:129)
transition from a quiescent vasculature to an Vasculogenesis (cid:129) Intussusception (cid:129)
actively growing one follows a series of syn- Vasculogenicmimicry(cid:129)Vesselgrowing(cid:129)Ves-
chronic events and is finely tuned by a wide sel guidance (cid:129) Vascular-remodeling factors (cid:129)
array of molecules and positive and negative Vessel stabilization (cid:129) Proangiogenic factors (cid:129)
regulators of angiogenesis. Beginning with Antiangiogenic factors (cid:129) Angiogenic switch (cid:129)
blood vessel sprouting and endothelial cell Hypoxia(cid:129)Tumorvasculature
proliferation, followed by vessel navigation,
remodeling,stabilization, andmaturation,and Contents
finishing with blood vessel regression, the
Introduction.......................................... 2
main molecular factors involved in the pro-
MolecularMechanismsofAngiogenesis........... 2
gression of each step are profoundly detailed.
MechanismsInvolvedinBloodVesselFormation... 2
Whenthebalancebetweenpositiveandnega-
BiologicalProcessesInvolvedinAngiogenesis...... 6
tive regulators of angiogenesis is shifted
TheAngiogenicSwitchinTumorigenesis.......... 18
towardproangiogenicmolecules,thequiescent
HypoxiaandTumorAngiogenesis................... 19
vasculaturebecomesactivatedandinitiatesthe DifferencesbetweenPhysiologicalandTumor
angiogenic state of tumor development. The Neovascularization................................ 21
role of intratumoral hypoxia as a potent
Conclusion............................................ 23
Cross-References..................................... 24
I.Zuazo-Gaztelu(cid:129)O.Casanovas(*) References............................................ 24
TumorAngiogenesisGroup,ProCURE,CatalanInstitute
ofOncology-IDIBELL,Barcelona,Spain
e-mail:[email protected];
[email protected]
#SpringerInternationalPublishingAG2016 1
D.Marmé(ed.),TumorAngiogenesis,
DOI10.1007/978-3-319-31215-6_1-1
2 I.Zuazo-GazteluandO.Casanovas
Introduction Molecular Mechanisms
of Angiogenesis
Tumorangiogenesis,theprocessbywhichblood
vesselspenetrateandgrowinthetumormicroen- MechanismsInvolvedinBloodVessel
vironment, is essential for oxygen and nutrient Formation
supply and, therefore, for survival of solid neo-
plasms. Taking into account its role in tumor Vessels can be described as highly branched and
progression and metastasis, angiogenesis is one ordered tubular networks that allow transport of
of the widely acknowledged hallmarks of cancer gases, nutrients, signaling molecules, and cells.
(Hanahan and Weinberg 2011). Since the Beyond their nutritive function, blood vessels
pioneering studies by Folkman established the provide instructive trophic signals essential for
initial foundations of tumor angiogenesis nearly organ morphogenesis and the development of
40 years ago, research in the field has reached a every complex organism (Carmeliet and Jain
significant level of maturity, allowing detailed 2011). While the luminal side of all types of
descriptionoftheintricateprocessesofpatholog- blood vessels, including arteries, veins, and cap-
icalvesselproliferation(Folkmanetal.1971).In illaries, is formed by a lined monolayer of endo-
this chapter, we compiled the basic mechanisms thelialcells,vesselsarecoveredintheoutsidebya
ofbloodvesselformationandthebiologicalroles basementmembranefollowedbyalayerofmural
of the main molecular regulators involved in accessory cells (pericytes and vascular smooth
tumorangiogenesis.Furthermore,wedescribein musclecells(SMCs)).
detailthe“angiogenicswitch”occurringinmalig- Prototypically, vasculogenesis and sprouting
nantneoplasms,focusingontheroleofthecom- angiogenesis are the two main mechanisms
plex tumor microenvironment, the differences responsible for neovascularization (Fig. 1a, b).
between physiological and pathological angio- Sprouting angiogenesis is defined as the forma-
genesis, and the abnormalities found in tumor tionofnewvascularstructuresfromapreexisting
vasculature. vessel, while vasculogenesis refers to de novo
Fig.1 Mechanismsinvolvedinbloodvesselformation. growth,aprocessthatinvolvesvesselsplitting(c).Addi-
Innormaltissuesvesselscangrowbytherecruitmentof tionally, tumor cells use other mechanisms such as
bonemarrow-derivedendothelialprogenitorcells(EPCs) vasculogenicmimicry,duringwhichvessel-likestructures
that differentiate into endothelial cells (a), by sprouting arelinedbytumorcells(d)
angiogenesis (b), or by intussusceptive microvascular
MechanismsofTumorAngiogenesis 3
bloodvesselformationduetovascularprogenitor homingtoneo-angiogenicspots areyet notfully
cell differentiation. Both mechanisms contribute understood.
to the formation and remodeling of the vessel MobilizationofEPCsstartsfollowingtheacti-
networkduringdevelopment,remainnearlyinac- vationofthematrixmetalloprotease9(MMP9)in
tiveintheadultbody,andareonlyreactivatedto the osteoblastic zone by the tumor-derived pro-
allowtissuerepairorintheeventofadisease. angiogenicfactors.TheactivationofMMP9trig-
Beyond vasculogenesis and sprouting angio- gers the proteolytic processing of membrane-
genesis, other less frequent mechanisms have bound Kit ligand to its soluble active form. The
recently been reported in neoplasms, including soluble Kit ligand is a stem cell-active cytokine
vessel co-option, intussusception, and that promotes migration of hematopoietic and
vasculogenicmimicry(Fig.1c,d).Inmostcases, endothelial progenitor cells to the vascular zone
mutualexclusivitybetweendifferentmechanisms ofthebonemarrowandtheirposteriorreleasein
does not exist; indeed, they simultaneously par- thecirculation(Heissigetal.2002).
ticipate both in physiological and pathological Whenhomed,endothelialprogenitorcellscan
angiogenesis. beincorporatedintotheendothelialmonolayerof
avesselorrecruitedtoangiogenicsprouts.Atthis
Vasculogenesis step,P-selectin,E-selectin,andintegrinsarecrit-
Vasculogenesis (Fig. 1a) has been extensively icalforthecorrectadhesionofEPCstothevessel
describedintheearlystagesofvasculardevelop- walls (Deb et al. 2004). Differentiation into
ment.Itwasnotuntil1997thatthegrowthofnew mature endothelial cells is mainly mediated by
blood vessels in postembryonic tissues was con- VEGF and physically contributes to vessel size
sidered to occur also through vasculogenesis since it increases the diameter of the vessel. In
(Asaharaetal.1997).Moreover,compellingevi- additiontothephysicalcontributionoftheEPCs
dence suggests that bone marrow-derived circu- tothenewlyformedvessels,EPCssupportangio-
lating endothelial progenitor cells (EPCs) genesis by a paracrine mechanism that includes
contribute to the induction and progression the release of proangiogenic factors in neo-
of postnatal neovascularization (Kopp et al. vascularizationsitesofthetumorstromaorische-
2006). EPCs, also known as angioblasts, are cir- mictissues(UrbichandDimmeler2004).
culating cells that express several endothelial Formal demonstration of the contribution of
lineage-specific markers such as CD34, CD31, vasculogenesis to tumor angiogenesis has been
VEGFR-2,andTie-2. achieved through the use of knockout mice for
TogetherwithEPCs,maturecirculatingendo- inhibitorsofdifferentiationfactors.Thesefactors
thelial cells derived from blood vessel renewal initiate the mobilization of bone marrow-derived
alsotakepartinadultvasculogenesis. Moreover, dendritic cells to angiogenic sites in the tumor.
inordertofacilitateincorporationofthosecircu- Genetic ablation of inhibitors of differentiation
latingendothelialadultandprogenitorcellsandto factors disrupted tumor vascularization and
sustain the stability of the nascent vasculature, blocked tumor growing as a consequence
hematopoietic stem and progenitor cells are of an impaired angiogenesis (Benezra et al.
recruited(Koppetal.2005).Severalchemokines, 2001). The restoration of the mobilization
cytokines,andgrowthfactorsthatareproducedin capabilityofbonemarrow-derivedcellsbytrans-
response to tissue ischemia and tumor growth plantation of wild-type bone marrow rescued
promote mobilization and recruitment of EPCs. tumor neovascularization and growth in these
For instance, tumor cells produce proangiogenic knockoutmice.
factors, such as VEGF, and cytokines (i.e., The contribution of vasculogenesis to tumor
stromal-derived factor-1) that recruit bone vessel formation ranges from 0.1 to 50%
marrow-deriveddendriticcellsandpromotetheir depending on the experimental cancer model
proliferation and differentiation. The precise and tumor type. Lymphomas and hematological
mechanismsgoverningmobilizationofprecursor tumors are more dependent on bone marrow-
cells from the bone marrow and their posterior derived dendritic cells in comparison to other
4 I.Zuazo-GazteluandO.Casanovas
tumors. The knowledge obtained from the (Ferrara et al. 2003). At this step, an immature
study of tumor vasculogenesis has sparkled the blood vessel is formed, and the opposite
development of new applications at the clinical mesenchymal-endothelial transition directs the
setting. The potential existing correlation reversal of the proliferative state of endothelial
between the levels of endothelial progenitor and cells to the previous resting state. In detail, the
circulating cells in blood and the outcome in return to quiescence is retrieved by synthesis of
patients undergoing an antiangiogenic treatment newbasementmembraneandpericyteandmural
couldbeusedasacellularbiomarkerformonitor- cell recruitment (Jain 2003). This latter step is
ing the response to antitumor therapy (Bertolini known as vessel maturation and is characterized
etal.2003). byalackoftumorangiogenesis.
Recently, the study of tumor vasculogenesis Recent studies have remarked the specializa-
hasshiftedfromitsroleinprimarytumorgrowth tion that endothelial cells undergo in order to
toward the study of its implication in dissemina- enrolltheangiogenic process.Toachievelocally
tion and metastasis. In addition to angiogenesis neededvascularpatterns,themultistepprocessof
activation, EPCs are able to promote metastatic sprouting angiogenesis requires functional spe-
growth by homing into metastatic sites prior to cialization of endothelial cells in the angiogenic
tumorcellarrival(Koppetal.2005).Thesponta- sprout together with vascular guidance cues that
neous secretion of SDF-1 by EPCs generates a allow regulation of the topological extension of
gradient that could promote the extravasation the forming vessel. The main group of signaling
and development of the pre-metastatic niche (Jin pathways essential for the initial morphogenetic
etal.2012). events includes VEGF and Notch (Iruela-Arispe
and Dvorak 1997). Attracted by proangiogenic
SproutingAngiogenesis signals, the phenotype of the sprouting endothe-
Sprouting angiogenesis is the best described lial cells gains an invasive and motile behavior,
mechanismusedbytumorstopromotetheirown protease activation, cell-cell contact remodeling,
vascularizationbyinducingnewcapillarysprouts andapical-basalpolarityreversal.Theendothelial
from preexisting host capillaries (Fig. 1b). The cells that are selected to guide the sprouting are
mechanisminvolvesseveralwell-definedsequen- locatedatthetipoftheangiogenicsproutandare,
tialstepsandanextensiveinterplaybetweensol- therefore,commonlyknownas“tipcells.”These
uble factors, extracellular matrix (ECM) leading cells respond to VEGF signaling by
components, and cells (Paku and Paweletz dynamicallyextendinglargefilopodiainorderto
1991). At the onset of sprouting angiogenesis, sense and guide the forming vessel along the
there is a destabilization of the endothelial- forthcoming vascular bed. Recent studies show
pericyte contacts which are essential for vessel similarities between the molecular regulation of
integrity and quiescence maintenance. Endothe- guidance cues of neural and endothelial cells.
lial and mural cells share a complex basement These specialized nonproliferative endothelial
membrane that forms a protective coat around cells also release different molecular signals that
endothelial tubules, preventing resident endothe- promote the recruitment of mural cells like peri-
lial cells from leaving their location. Once cytes, SMCs, and fibroblasts, guaranteeing the
destabilized, endothelial cells undergo an stabilizationofemergingvessels.
endothelial-mesenchymaltransitionthatenhances Apartfromtipcells,endothelialcellscanspe-
their migratory, invasive, and proliferative prop- cialize to highly proliferating cells located in the
erties. These activated cells are then able to stalk of the angiogenic sprout. The proliferative
degrade the surrounding ECM and the basement potential of the “stalk cells,” capable of forming
membrane by activated proteases (such as tubes and branches, assures the expansion of
MMPs), opening the path for guided migration the structure. In contrast to tip cells, stalk
and proliferation. Vessel lumen is then formed cellsextendfewerfilopodiabutproliferatetosup-
by polarization of themigrating endothelial cells port vessel elongation in response to VEGF-A
MechanismsofTumorAngiogenesis 5
(Gerhardtetal.2003).Tipcellsaremigratoryand tumor angiogenesis, there are alternative pro-
polarized, whereas stalk cells proliferate during cesses such as intussusceptive microvascular
the extension of the sprout and form the new growth(IMG;Fig.1c)andvasculogenicmimicry
vascular lumen cell population. Stalk cells also (VM; Fig. 1d). These nonconventional pathways
produce components of the basement membrane introduceanadditionallevelofcomplexitytothe
and establish adherens and tight junctions with understanding of tumor vascularization
neighboringcells,thusstrengtheningtheintegrity mechanisms.
ofthenewsproutandluminal-abluminalpolarity
(Dejanaetal.2009). Intussusception
Inordertoformanewvascularconnection,the Intussusception(IMG)isa variant ofangiogene-
tip cell phenotype must be switched off after sisthatwasfirstobservedinpostnatalremodeling
connecting with the tips of other sprouts or of lung capillaries (Caduff et al. 1986; Fig. 1c).
existing vessels. Tip cells build vessel loops by This developmental intravasculargrowthmecha-
anastomosing with cells from neighboring nism is based on the splitting of preexisting ves-
sprouts. The sprouting process is rehearsed until selsintotwonewvesselsaftertheformationofa
proangiogenic signals decrease, a new basement transvascularconnectivetissuecolumn,calledtis-
membraneisformed,quiescenceisreestablished, suepillar,intothelumenofthevessel.
andVEGFlevelsdampen(Leslieetal.2007).In Incontrasttosproutingangiogenesis,IMGisa
thetransitionfromactivesproutingtoquiescence, fast process that can occur within hours, or even
endothelial tip cells adopt a “phalanx”-like phe- minutes,sinceitdoesnotrequireproliferationof
notype, with features of lumenized, non- endothelial cells. Even though sprouting has the
proliferative, and immobile cells (Bautch 2009). advantage of being invasive and permits joining
Eventually, maturity and stabilization are vascular gaps, it is a slow process that highly
achieved through the generation of a lumen and relies on endothelial cell proliferation and basal
the migration of pericytes along the basement membrane degradation. In IMG, the remodeling
membrane until vessels are covered, initiating ofendothelialcellsisaconsequenceoftheirvol-
bloodflowandallowingperfusion. ume increase and narrowing. It is believed that
The correct extension and morphology of the IMG happens after vasculogenesis or sprouting
nascentvesselsisregulatedbytheprecariousbal- angiogenesis in order to expand the capillary
ance between tip cell navigation and stalk cell plexus without a high metabolic demand (Burri
proliferation. The phenotypic specialization to etal.2004).
tip or stalk cell depends on the balance between TheonsetofIMGisthe“touchingcontact”of
proangiogenic factors andendothelialcellprolif- endothelial cells from opposite walls. Following
erationsuppressors(GeudensandGerhardt2011). the transendothelial cell bridge created from the
Theabnormalvascularstructuresgenerallyfound touching spot, interendothelial junctions are
in cancer are a consequence of the imbalance reorganized, and the endothelial bilayer is
between these two processes. The biological performed. Then, the interstitial pillar forms to
nature of the molecules and signals that initiate reinforcethebridges,andmuralcellsarerecruited
the angiogenic cascade from the initial destabili- to cover this new interstitial wall. Due to their
zation to the formation of mature and functional contractile features, pericytes are believed to be
vasculaturehasbeenprofoundlystudiedandchar- themaintriggersofthisphase.Finally,theinter-
acterizedandisfurtherdescribedinthefollowing stitial pillars widen, endothelial cells retract, and
sectionsofthischapter. two independent vessels are created (Burri et al.
2004). By IMG, a large vessel can split into
AlternativeWaysofBloodSupply smallervessels.
inTumors Even though the precise mechanism of intus-
Although sprouting angiogenesis is regarded as susception is poorly understood, there are some
the most important contributing mechanism to key mediators that influence pillar formation.
6 I.Zuazo-GazteluandO.Casanovas
Alterations in blood flow dynamics, changes in components, and low levels of oxygen are
shearstressonendothelialcellssensedandtrans- known to promote VM (Seftor et al. 2005).
ducedbymoleculessuchasCD31,modifications Although the exact mechanism remains to be
of wall stress on the pericytes, and absences of unraveled, it involves deregulation of the
VEGFaresomeamongthepossiblefactorsdriv- lineage-specific phenotype and the concomitant
ingbiochemicalcascadesthatresultincytoskele- transdifferentiationtoendothelial-likecells.
tal rearrangements and intussusception initiation VM occurs mainly in aggressive tumors such
(DjonovandMakanya2005).Forinstance,itwas asmelanomas,and even though theiroccurrence
observedthathumanmelanomasbareahighnum- is relatively rare within tumors, the presence of
berofintraluminaltissuefoldstogetherandthata VM-associatedpatternednetworksintumortissue
correlation between VEGF and intussusceptive correlateswithanincreasedriskofmetastasisand
angiogenesisexistsinthesetumors(Ribattietal. poor clinical outcome (Sun et al. 2004). Until
2005). In this context, sprouting angiogenesis now, VM has been described in melanomas,
inhibitionmightstimulatetheprocessofintussus- breastcarcinoma,prostaticcarcinoma,hepatocel-
ceptive angiogenesis. Since IMG can only occur lular carcinoma, bladder carcinoma, and other
on existing vessel networks, its most important aggressive tumors. Increasing evidence demon-
contribution is its ability to increase the density strates that tumor cell-dominant VM has a key
and complexity of tumor microvessel networks roleintumorprogressionandmetastasis.
already established by sprouting angiogenesis.
Moreover, IMG also provides additional surface
forfurthersproutingangiogenesis.IMGhasbeen BiologicalProcessesInvolved
observedincolorectal,melanoma,andmammary inAngiogenesis
tumors(Domeetal.2007).
As stated above, in order to build new and fully
VasculogenicMimicry functional vascular structures, several biological
Vasculogenic mimicry (VM; Fig. 1d) describes processesmustbeaccuratelyregulated.Different
the ability of some tumor cells to dedifferentiate pro- and antiangiogenic regulators are needed to
into multiple cellular phenotypes, obtaining perform and control each specific step of the
endothelial-likeproperties(Maniotisetal.1999). angiogenic cascade. Multifunctionality among
This process leads to the creation of de novo those factors is one of their relatively common
vasculogenic-like matrix embedded networks. features, empowering some molecules with the
The new perfusable vascular-like structures are outstanding ability to either activate or inhibit
composedofredbloodcellsandplasmaandcon- vascularization.Onaccountofsimplification,we
tribute to blood circulation (Frenkel et al. 2008). willdescribethemolecularregulationofthemain
Endothelial cells undergoing VM mimic the pat- biologicalprocessesinvolvedinvesselformation:
ternofembryonicvascularnetwork,possiblypro- sprouting and proliferation (Fig. 2a), guidance
viding tumor cells with a secondary circulation and navigation (Fig. 2b), stabilization, matura-
system,independentfromangiogenesis. tion,andremodeling(Fig.2c),andregression.
Molecularanalysiscomparinghighlyinvasive
andnoninvasivemelanomacellsderivedfromthe SproutingofBloodVessels
same patient suggests a genetic reversion of andEndothelialCellProliferation
the aggressive cells to an embryonic-like cell Endothelial cells in the adult organism remain
fate and increased cell plasticity. The quiescent and are protected against external
undifferentiated phenotype includes the expres- insults by autocrine maintenance signals. These
sion of endothelium-associated genes such as cells form a monolayer of phalanx cells and
VE-cadherin and Ephrin-A2, among others are interconnected by junctional molecules like
(Hendrixetal.2003).Theactivationoftransmem- VE-cadherin and claudins. The surface of
brane metalloproteinases, release of ECM the endothelium monolayer is covered by
MechanismsofTumorAngiogenesis 7
Fig. 2 Molecular basis of angiogenesis. Sequential pericyte detachment. A provisional matrix layer is
steps of blood vessel formation and their features are depositedbyextravasationofplasmaproteins(e.g.,fibrin-
depicted.Themostimportantmolecularplayersinvolved ogen) due to increased permeability. Cell migration is
ineachprocessaredenotedinparentheses.(a)Uponangio- favoredbyprotease-mediatedmatrixremodeling.(b)Tip
genicstimulationbyproangiogenicfactors,thequiescent cells sense the environment and navigate in response
vessel dilates and an endothelial tip cell is selected. to guidance cues (e.g., semaphorins and ephrins) while
Tipcellgenerationrequiresbasementmembranedegrada- adhering to the extracellular matrix (ECM) in order to
tion, loosening of endothelial cell-cell junctions, and migrate.Stalkcellsbehindtheleadingtipcellproliferate
8 I.Zuazo-GazteluandO.Casanovas
pericytes, which suppress endothelial cell prolif- hypoxia,oncogeneactivation,lossoftumorsup-
eration, and releases pro-survival signals such as pressors, cytokines, and growth factors levels.
VEGFandAng-1.Whenquiescentvesselssense There is also an autocrine production of VEGF
an angiogenic signal such as VEGF, fibroblast by endothelial cells which is critical for vascular
growth factors (FGFs), or chemokines, released homeostasisandearlystagesofvasculardevelop-
by hypoxic, inflammatory, or tumor cells, ment(Leeetal.2007).Ingeneralterms,paracrine
sproutingangiogenesisistriggered(Fig.2a). VEGF, released by tumor or stromal cells,
VEGF-VEGFR signaling pathway is increases vessel branching and promotes tumor
established as the master regulator of the forma- vessel abnormalities (Stockmann et al. 2008),
tion and remodeling of vasculature. VEGF whereasautocrineVEGF,releasedbyendothelial
ligands are the prototypical, multifunctional pro- cells, empowers vascular homeostasis. The dele-
angiogenic factors that control endothelial cell tion of a single allele of VEGF-A causes embry-
proliferation and migration and regulate cardio- oniclethality,reinforcingthebelievedkeyroleof
vascularsystemhomeostasis(Carmeliet andJain thisfamilyindevelopmentalvascularphysiology.
2011).Untilnow,VEGFmoleculesareallegedly There are three different tyrosine kinase-type
themostpotentvascularpermeabilityfactorsand receptorsfortheVEGFfamily:VEGFR-1(orFlt1
vasodilatationinductors.Besides,endothelialpre- inmouse),VEGFR-2(alsoknownaskinaseinsert
cursor cell differentiation and vascular guidance domain-containing receptor in humans or Flk1),
oftipcellsarealsocontrolledbyVEGFfamily. andVEGFR-3(orFlt4).Thesereceptorsareprin-
TheVEGFfamilyiscomposedofsixdifferent cipally expressed in endothelial cells (Roskoski
members: VEGF-A (referred herein as VEGF), 2008). Furthermore, certain VEGF isoforms are
VEGF-B,VEGF-C,VEGF-D,VEGF-E,andpla- also able to bind to non-tyrosine kinase
cental growth factor (PlGF). Contrary to other coreceptorssuchasneuropilins1and2toenhance
angiogenic superfamilies, the VEGF family dis- VEGFR-2 activity. Neuropilins are best known
tinguishes itself by the nonredundant role of its for their interaction with semaphorin and their
members. At least five different isoforms of angiogenesis-independent function in axonal
VEGF are generated by alternative splicing of a guidance(Gluzman-Poltoraketal.2000).
single gene: VEGF , VEGF , VEGF , VEGFR-1hasbeenidentifiedasthehighaffin-
121 145 165
VEGF , and VEGF . These isoforms differ ityreceptor,whereasVEGFR-2isthelow-affinity
189 206
intheirbindingaffinityforheparin,whichmight receptor. While placental growth factor and
affect their diffusion rates in the extracellular VEGF-B bind to VEGFR-1, VEGF-A can bind
space. The predominant splicing variant of both VEGFR-1 and VEGFR-2, VEGF-C and
VEGF both in normal and tumor cells is the VEGF-D bind to VEGFR-2 and VEGFR-3, and
VEGF isoform.AllthemembersoftheVEGF VEGF-E binds to VEGFR-2. Binding of the
165
family and their corresponding receptors usually ligand leads to the dimerization of the receptors;
work as homodimers, although heterodimers this in turn initiates the autophosphorylation of
between different members have also been several intracellularly located tyrosine residues.
reported(DiSalvoetal.1995). The activated dimers now expose new docking
During tumor progression, VEGF levels are sitesfortherecruitmentofdifferenttypesofinter-
controlled through diverse mechanisms such as mediary signaling molecules by protein-protein
(cid:1)(cid:1)(cid:1)
Fig.2 (continued)andelongate,attractingpericytesinthe posterior lumen formation drive neovessel perfusion,
process.Stalkcellsarefurtherstabilizedbythedeposition whichconcludeswithquiescencebyaphalanxphenotype
of the new basement membrane. Immune myeloid cell promotion, deposition of the new basement membrane,
recruitment of tumor-associated macrophages (TAMs) maturationofpericytes,reestablishmentofcell-celljunc-
andTIE-2-expressingmonocytes(TEMs)producesaddi- tions,andreleaseofvascularmaintenancemolecules
tional proangiogenic factors and triggers the release of
ECM-boundfactors.(c)Fusionofadjacentbranchesand
MechanismsofTumorAngiogenesis 9
interactions through specific SH2 and SH3 shown that VEGFR-1 acts as a decoy receptor,
domains. These large signaling complexes are since it is able to sequester VEGF, implying a
known as signalosomes and can be different negativeroleforthisreceptorinangiogenesis.In
depending on the combination of VEGF ligand addition,micelackingVEGFR-1presentahigher
and receptor. In fact, differences between number of endothelial cells than wild-type mice,
signalosomes allow a broad range of biological whereas the decreased expression of VEGFR-1
effects to VEGF stimulation that include increases VEGF availability and VEGFR-2
increasedendothelialcellproliferation,migration, activity. In fact, a soluble isoform of VEGFR-1
survival,permeability,andECMdegradation. (sVEGFR-1), which encodes the extracellular
Several studiesindicate that VEGF stimulates ligand-binding domain, can be produced by sur-
both physiological and tumor angiogenesis by rounding cells. While soluble VEGF isoforms
signalingthroughVEGFR-2inadose-dependent promote vessel enlargement, matrix-bound
manner (Carmeliet and Jain 2011). In fact, isoforms stimulate the branching pattern.
VEGFR-2nullmicedieduringembryonicdevel- sVEGFR-1 inhibits angiogenesis by acting as a
opment by defects in the vascular system, molecular trap for VEGF ligand, assisting the
reinforcing its role in proliferation, survival, and guidance of emerging branches or inhibiting the
migration of vascular endothelial cells. The acti- sprouting. The imbalance between the functions
vation of VEGFR-2 promotes differentiation of ofVEGFR-1andVEGFR-2causeshemangiomas
progenitors, mitogenesis, chemotaxis, survival, andbenigntumorswithincreasedaberrantangio-
and vascular permeability. It also increases the genesis(Jinninetal.2008).
expressionofmatrixmetalloproteinasesandplas- Inpathologicalconditions,PlGFcontributesto
minogenactivatorsforECMdegradationandfur- the angiogenic switch by affecting multiple cell
ther endothelial cell migration. In detail, VEGF type directly and indirectly and also activates
release causes plasma proteins extravasation and bone marrow-derived endothelial progenitor
the deposition of a provisional ECM scaffold cells. PlGF can induce its own signaling and
toward which endothelial cells migrate in amplify VEGF-driven angiogenesis through
responsetointegrinsignaling.Theactivatedpro- direct effects on endothelial cells (Autiero et al.
teasesliberatetheangiogenicmoleculesstoredin 2003).ThesynergismbetweenVEGFandPlGFis
theECMsuchasVEGFandFGF,andtheECMis also stated by the fact that PlGF upregulates the
remodeledintoanangio-competentmilieu.Once expression of VEGF. PlGF might also indirectly
VEGFisreleased,itbindstotheVEGFR-2recep- influence SMC proliferation and migration
tors oftheendothelial cells.Tipcellmigration is throughactivatedendothelialcellcytokinerelease
regulated by VEGF gradient, whereas stalk cell (Luttunetal.2002).
proliferation depends on VEGF concentration Other prototypical proangiogenic signaling
(Gerhardtetal.2003).VEGF/VEGFR-2signaling pathway involves the fibroblast growth factor
axis induces the formation and extension of (FGF) family, comprised by 23 different ligands
filopodia and the expression of delta-like ligand and four tyrosine kinase-type receptors (FGFR-
4(Dll4)proteinintipcells,whichactivatesNotch 1–4) expressed widely in the organism (Presta
in stalk cells. Notch, in turn, downregulates et al. 2005). FGFR-1 and FGFR-2 are expressed
VEGFR-2 expression in stalk cells, rendering by endothelial cells, and their binding to ligands
them less responsive to VEGF and ensuring tip FGF-1, FGF-2, FGF-4, and FGF-5 leads to the
cellleading.TheblockadeofVEGFR2signaling induction of critical stages of angiogenesis
isassociatedwithsproutingdefects(Bentleyetal. in vivo. Among the four pleiotropic pro-
2009). angiogenic ligands, at least FGF-1 and FGF-2
On the other hand, VEGFR-1 is only slightly directly stimulate endothelial cell proliferation,
activatedbyproangiogenicfactors,anditsprecise detachment, migration, and ultimate differentia-
role in angiogenesis is poorly understood tion into a functional capillary vessel. For the
(Schwartz et al. 2010). Nevertheless, it has been maintenance of vascular integrity, endothelial
10 I.Zuazo-GazteluandO.Casanovas
quiescent cells require low levels of FGF, since like TGF-β induce the autocrine production of
vesseldisintegrationhasbeenobservedasacon- EGF-likemoleculessuchasTGF-α,thuspromot-
sequence of FGFR signaling inhibition ing endothelial cell survival through PI3K-Akt
(Murakami et al. 2008). FGF ligands exert their signaling(ViñalsandPouyssegur2001).
functions in endothelial cells after paracrine The ECM itself provides a link between vas-
release by stromal or tumor cells, or by endoge- cular cells and their surrounding environment.
nousFGFinanautocrinefashion. Proteolytic degradation of the basement mem-
Even though VEGF has a pivotal role during brane and the surrounding ECM is an integral
angiogenesis, an important cross talk takes place partofangiogenesis.Inthisstep,severalprotein-
between FGF and VEGF. For instance, VEGF ase families are involved, including matrix
systemactivationisrequiredforlaterFGFinduc- metalloproteinases (MMP and their tissue-type
tion and in vivo angiogenesis promotion. The inhibitors or TIMP), plasminogen activators
opposite cross regulation has also been demon- (uPA and its inhibitor PAI-1), heparanases,
strated, and FGF also seems able to stimulate tryptases, chymases, cathepsins, etc. Besides
tumor angiogenesis under certain experimental breaking down ECM components and clearing a
conditions.TheeffectsofFGFareduetoitsdual pathforendothelialcellmigration,proteinasesare
action including a direct effect over endothelial abletoswitchonangiogenesisbytheliberationof
cellsandanindirecteffectconcerningtheregula- matrix-bound angiogenic activators (bFGF,
tion of the production of other proangiogenic VEGF, TGF-β, HGH, etc.) and proteolytically
molecules like VEGF, angiopoietin-2 (Ang-2), activatingangiogenicchemokinessuchasIL-1β.
or interleukin-8 (IL-8) by tumor or stromal cells WhereasVEGFisoformscleavedbyMMPspref-
(BeenkenandMohammadi2009).Bothinmouse erentiallyenlargevessels,MMP-resistantmatrix-
and human tumors, the role of FGF in tumor bound VEGF is involved in vessel branching
growthandneovascularizationhasbeendescribed (Iruela-Arispe and Davis 2009). Moreover, pro-
(Prestaetal.2005). teasessuchasMMP9participateinthemobiliza-
Thethirdproangiogenicpathwayincludesthe tionofbonemarrowprogenitorsbytheliberation
epidermal growth factor (EGF)-like family of ofcytokinessuchastheKitligand(Heissigetal.
growth factors and their receptors (ErbB). The 2002)andbytheestablishmentofapre-metastatic
integrants of this family play various functions niche (Kaplan et al. 2005). The proteolytic
indifferenttissues,buttheyarebasicallyinvolved remodeling of the ECM occurs in a sharply con-
in cell proliferation and survival stimulation. trolledmode,andthepleiotropicactivitiesofpro-
SomeoftheligandsincludedareEGFandtrans- teinasesarecontextandconcentrationdependent.
forminggrowthfactoralpha(TGF-α),whichbind In fact, excessive breakdown removes guidance
to the ErbB family of tyrosine kinase receptors. cuesforendothelialcellmigration,thusinhibiting
The ErbB family of receptors is composed angiogenesis, while insufficient degradation pre-
of ErbB1/HER1/EGFR, ErbB2/HER2/Neu, vents vascular cell mobility (Carmeliet and Jain
ErbB3/HER3,andErbB4/HER418. 2011).
Bothintumorandphysiologicalsettings,EGF Moreover, proteinases can switch off angio-
family members display proangiogenic activity. genesis,astheyliberatematrix-boundangiogenic
However, whether the effects of EGF are direct inhibitorssuchasarrestin,angiostatin,TSP-1,and
orindirectremainsunclear.EGFstimulationpro- inactivate angiogenic cytokines like SDF-1. The
ducesthereleaseofproangiogenicfactorsuchas basementmembraneofquiescentvesselsiscom-
VEGF,IL-8,andFGFbytumorandstromalcells. posedmainlyofcollagenIVandlaminin,whereas
The ErbB family receptors in endothelial cell the interstitial matrix of collagen I and elastin
membranes enable these cells to respond to betweenvascularcellsprovidesfurtherviscoelas-
EGF-like factors by increased proliferation and ticity and strength to the endothelial cell wall.
survival. Furthermore, other angiogenic factors Proteinases expose novel epitopes of these ECM