Angiogenesis – background
lecture I
5th March 2012
AngiogenesisAngiogenesis
� An�gi�o�gen�e����sis (an-jee-o-JEN-uh-sis):
angei-, angeio-, vessel or blood vessel,
genesis-, origin or birth
angiogenesis:
formation of new blood vessels via
extension or remodeling from existing
capillaries
CirculatoryCirculatory system system andand bloodblood vesselsvessels
BloodBlood vesselsvessels
Cleaver O & Melton DA, Nature Med., June 2003
pericytes
pericytes
pericytes
CapillariesCapillaries withwith pericytespericytes
Examples of vascular defectsExamples of vascular defects
TheThe vascularvascular wallwall
vascular wall
remodeling
EndotheliumEndothelium regulatesregulates
vascular tone
thrombosis
fibrinolysis
inflammation
angiogenesis
Tousoulis, et al., Heart 2005;
Endothelial cell functionsEndothelial cell functions
• Maintaining the vascular tone: Vasodilation and Vasoconstriction
BloodBlood vesselvessel formationformation -- shortshort historyhistory ofof studiesstudies
• Roman physician Claudius Galen of Pergamon (129–199) found
that vessels are filled with blood. But he claimed that blood is
produced in the liver.
• Then, sir William Harvey (1578 -1657) discovered the circulation
of blood
• Harvey proved that blood flows around the body. He stated that
blood was carried away from the heart by arteries and returns to
the heart through veins.
•• Harvey proved that the heart is a pump
that recirculates the blood
William Harvey - 1628
the first systematic description of circulatory system
(1578-1657)
HarveyHarvey’’s Methodss Methods
Harvey was very scientific in his methods. These included:
• Dissecting cold blooded animals (e.g. reptiles) to observe the movement of muscle around the heart.
• Dissecting humans to gain a knowledge of the heart.
• He accurately calculated the amount of blood in the body.
• The word „angiogenesis” was used for the first time in 1787 by
british surgeon, Dr. John Hunter, to describe the growth of blood
vessels in reindeer antler.
BloodBlood vesselvessel formationformation -- shortshort historyhistory ofof studiesstudies
• The term „endothelium” was introduced by Wilhelm His in
1865 to differentiate the inner lining of body cavities from
„epithelium”
• In 1661 Marcello Malpighi observed capillaries using
chicken embryo model
Die Häute und Höhlen des Körpers.
Basel, Schwighauser, 1865.
A new classification of tissues based on histogenesis. In the present work, His put forth the basic concepts of
tissue embryology. Using serial sections and three-dimensional models to illustrate his theories, he showed that
the serous spaces in the embryo are mesodermal in origin and that they are lined by the special layer which he
was the first to term "endothelial"
• End of XIX/begining XX century Henryk Hoyer –
(Jagiellonian University) – description of lymphatic system
• Detailed study on vasculogenesis in chick embryo and
lymphangiogenesis by Florence Sabin
BloodBlood vesselvessel formationformation -- shortshort historyhistory ofof studiesstudies
Diagram of the organization of lymphatic and
vein systems in chordate
3, dogfish; 4, fish; 5, newt and salamander; 6,
frog and toad; 7, lizard; 8, crocodile; 9, bird;
10, mammals.
Distribution of the surface lymphatic
veins on the head of trout (Salmo
trutta).
BloodBlood vesselvessel formationformation -- shortshort historyhistory ofof studiesstudies
• Gimbrone MA Jr, Cotran RS, Folkman J.
Endothelial regeneration: studies with human endothelial cells in culture.
Ser Haematol. 1973;6(4):453-5
• Jaffe EA, Nachman RL, Becker CG, Minick CR
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and
immunologic criteria.
J Clin Invest. 1973 Nov;52(11):2745-56
Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical
vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for
10 serial passages.…..
• Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, Lawley TJ.
HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest
Dermatol. 1992 Dec;99(6):683-90.
Model Model organismsorganisms inin vascularvascular researchresearch
Transgenic zebrafish allowTransgenic zebrafish allowss analysis of analysis of
endothelial cells in living embryosendothelial cells in living embryos
Dorsal AortaDorsal Aorta(DA)(DA)
Posterior Cardinal Vein(PCV)
Intersegmental VesselsIntersegmental Vessels(Se)(Se)
Dorsal Longitudinal Anastomotic VesselDorsal Longitudinal Anastomotic Vessel(DLAV)(DLAV)
Caudal Vein Caudal Vein Capillary PlexusCapillary Plexus
VEGFVEGF isis required for vasculogenesis in zebrafishrequired for vasculogenesis in zebrafish
Nasevicius et al., 2000
Microangiography allows high resolution mapping of mature vessels
EndothelialEndothelial cells cells
The crucial player in blood vessel formation
Endothelial cells in culture – cobblestone appearance
Human microvascular endothelial cells (HMEC-1)
• main players in angiogenesis
• immortalized cell line
• generate detectable amount of VEGF
• extended life span
Human umbilical vein endothelial cell line (HUVEC)
• macrovascular cells
• produce very small amount of VEGF
• dependent on growth factors
• limited life span
VariousVarious typestypes ofof endothelialendothelial cells cells
IsolationIsolation ofof HUVEC HUVEC cellscells
Endoglin is an auxiliary receptor for the transforming growth factor-beta family of cytokines
and is required for angiogenesis and heart development.
vWF - Von Willebrand factor
QuiescentQuiescent andand angiogenic angiogenic endothelialendothelial cellscells
Endothelial cell markers used to identify Endothelial cell markers used to identify
microvasculature in tissuesmicrovasculature in tissues
Scavenger receptorUptake of Ac-LDL
Factor VIII von Willebrand Factor (vWF)
VEGFVEGFR-2
AngiopoietinsTie-2
Ephrin B2EphB4
EphB4Ephrin B2
ET-1Endothelin receptor B (ETBR)
CD144 homotypic interactionCD144 (VE-cadherin)
ThrombinCD141 (thrombomodulin)
VLA-4 integrinCD106 (VCAM-1)
TGF-β1 and –β3CD105 (endoglin)
Sialyl-Lewis-X antigen and other carbohydratesCD62E (E-selectin)
LFA-1 integrinCD54 (ICAM-1)
L-selectinCD34
CD31 on endothelial cells, leukocytes; glycosaminoglycansCD31 (PECAM-1)
LigandMarker
kidney small intestine liver
Cells in an organ under the control of Cells in an organ under the control of
organ specific influencesorgan specific influences
MorphologicalMorphological differentiationdifferentiation ofof capillariescapillaries
Skeletal muscle, heart,
lung, brainEndocrine and exocrine organs
Cleaver O & Melton DA, Nature Med., June 2003
CONTINUOUS =
UNFENESTRATEDBasement
membrane
Endothelial
cellJunctions
Occluding
Gap
Adherens
Vesicles
ContinousContinous capillariescapillaries
Fenestrae/pore
80-100 nm
Endothelial
cell
Basement
membrane
Vesicles /
caveolae
FenestratedFenestrated capillariescapillaries
Endothelial cell
DiscontinousDiscontinous capillariescapillaries
Lack of
basement
membrane !
* sinusoids
Basal lamina
Endothelial
cellJunctions
Caveolae &
Vesicles
2. TRANSCYTOSIS
3. INTER-CELLULAR
PASSAGE
4. MEMBRANE
TRANSPORTERS
1. DIFFUSION
ContinousContinous capillariescapillaries –– transporttransport
Typical for
muscle & skin
fenestrae/pore 4. MEMBRANE
TRANSPORTERS
3. INTER-
CELLULAR
PASSAGE ?
2. TRANSCYTOSIS
1. DIFFUSION
5. THROUGH
PORES
Typical for gut, kidney,
& endocrine glands
Fenestrated capillaries – transport
HowHow thethe bloodblood vesselsvessels areare formedformed??
Susuma Nishinaga/Science Photo Library
Blood vessels in the small intestine
BloodBlood vesselvessel formationformation
a) vasculogenesis:
de novo blood vessel generation from
vascular progenitor cells
b) angiogenesis:
formation of new blood vessels via extension
or remodeling of existing blood vessels
c) arteriogenesis:
maturation of blood vessels via increasing
the lumen of vessels
BloodBlood vesselvessel formationformation
• Vasculogenesis:
a) during embryonic development;
• Angiogenesis:
a) embryonic development
b) adulthood: wound healing, menstrual
cycle, tumor-angiogenesis…
b) during adulthood associated with
circulating progenitor cells
PhysiologicalPhysiological angiogenesisangiogenesis inin adultsadults isis restrictedrestricted
placenta uterus
Hair growthWound healing
New New capillarycapillary formationformation inin responseresponse to to woundingwounding
AngiogenesisAngiogenesis maymay be be impairedimpaired inin many many diseasesdiseases
CardiovascularCardiovascular
diseasesdiseases
angioblastcapillary
bFGF
VEGF
VEGF
Ang1, bFGF
Vasculogenesiscapillaries are formed from
vascular progenitor cells
Angiogenesisformation of new blood vessels
from pre-existing vessels
Arteriogenesisformation of mature blood
vessels; differentiation into
veins and arteries
ThreeThree waysways ofof formationformation ofof bloodblood vesselsvessels
MCP-1, PDGF
Ang-2
Major Major growthgrowth factorsfactors andand receptorsreceptors involvedinvolved
inin bloodblood vesselsvessels formationformation
VEGF – vascular endothelial growth factors
VEGF-A – crucial mediator of angiogenesis
VEGF-R – receptors for vascular endothelial growth factors
Angiopoietins (Ang-1, 2)
Tie- 2 – receptor for Ang-1, -2
FGFs – fibroblast growth factors
PDGF – platelet-derived growth factor
Angiogenesis
• Majority of vascular development occurs
via angiogenesis
• Growth of new blood vessels from
existing vessels
• Two distinct mechanisms available
a) sprouting angiogenesis
b) intussusceptive angiogenesis
SproutingSprouting angiogenesisangiogenesis
• Sprouting: invasion of new capillaries into
unvascularized tissue from existing mature
vasculature
- degradation of matrix proteins
- detachment and migration of ECs
- proliferation
SproutingSprouting angiogenesisangiogenesis
IntussusceptiveIntussusceptive oror non non sproutingsprouting angiogenesisangiogenesis
- remodelling of existing vessels
- interendothelial contact is needed
- splits into two vessels
a: Remodeling: A pillar appears in closeproximity of the branching angle
b,c: From the pillar a fold developstoward the tissue of the branching angleand finally the pillar tissue merges withthe interstitium of the branching angle
d: As a result, the branching angle isrelocated proximalad and the angle isaltered
e: Pruning: A column of pillars arisesclose to the branching angleThe pillars can merge along a lineindicated by the arrows. The eccentriclocation of the pillars can lead to alterations in the diameter of a vascularbranch.
f: Ultimately, a branch can be completelypruned by repetition of the process(arrows along a theoretical line).
Scanning electron micrographs of chicken chorioallantoic membrane (CAM) vessels showing intussusceptive branching remodeling:
Burri, PH Dev Dyn. 2004
?
StagesStages ofof angiogenesisangiogenesis
Angiogenesis is a dynamic and context determined processAngiogenesis is a dynamic and context determined process
VascularVascular
EndothelialEndothelial
GrowthGrowth
FactorFactor
VV
EE
GG
FF
VascularVascular
PermeabilityPermeability
FactorFactor
VV
PP
FF
1983,1983,
Dr H. Dr H. DvorakDvorak
1989, 1989,
Dr N. FerraraDr N. Ferrara
Dr J. Dr J. PlouetPlouet
=
vascularvascular permeabilitypermeability factorfactor
endothelialendothelial cellcell survivalsurvival factorfactor
endothelialendothelial cellcell proliferationproliferation
endothelialendothelial cellcell migrationmigration
MainMain proangiogenicproangiogenic factorfactor
StagesStages ofof angiogenesisangiogenesis
1. increase in vessel permeability
2. loosening of pericyte contact
3. proteinase release from endothelial cells
4. digestion of basement membrane and extracellular matrix
5. migration and proliferation of endothelial cells
6. formation of vascular structures
7. fusion of new vessels
8. initiation of blood flow
inhibition of endothelial cell proliferation
inhibition of the migration of endothelial cells
9. formation of basement membrane
Angiogenesis Angiogenesis -- Basement Membrane BreakdownBasement Membrane Breakdown
Smooth Muscle
Cells
Endothelium
Basement
Membrane
Matrix
metalloproteinases
Angiogenic
Stimulus
(VEGF)
CrucialCrucial role role ofof metalloproteinasesmetalloproteinases inin angiogenesisangiogenesis
MMP-2 – gelatinase AMMP-9 – gelatinase B
elastinmetalloelastase (MMP-12)
type V collagenEnamelysin (MMP-20)
gelatinMMP-19 (RASI-1)Others MMP (examples)
type IV, V collagen, gelatinGelatinase B (MMP-9)
type I, IV, V and fibrillar
collagens; gelatin
Gelatinase A (MMP-2)Gelatinases
MT4-MMP (MMP-17)
gelatinase AMT3-MMP (MMP-16)
gelatinase AMT2-MMP (MMP-15)
gelatinase A, fibrillar
collagens, proteoglycans,
ECM glycoproteins
MT1-MMP (MMP-14)The membrane-bound
MMPs
MMP-26 (Matrilysin-2, endometase)
laminin, fibronectin, non-fibrillar collagensMatrilysin (MMP-7) PUMPMatrilysins
Stromelysin-3 (MMP-11)
Stromelysin-2 (MMP-10)
laminin, fibronectin,
non-fibrillar collagens
Stromelysin-1 (MMP-3)Stromelysins
MMP-18 (Collagenase 4, xenopus collagenase)
Collagenase-3 (MMP-13)
Neutrophil Collagenase (MMP-8)
fibrillar collagensInterstitial Collagenase (MMP-1)Collagenases
ECM SubstrateMMP NameMMP family
MatrixMatrix metalloproteinasesmetalloproteinases
Prodomain Catalytic domain Hemopexin
PRCGxPD
gelatin
binding
Zn2+
Zn2+
Zn2+
Zn2+
Zn2+
Collagenase family
Gelatinase family
MT-MMPs
MMP-7, MMP-23,
MMP-26
Hinge
region
Stromelysin family
Transmembrane
MatrixMatrix metalloproteinasesmetalloproteinases
MMPsMMPs areare pro pro andand antianti--angiogenicangiogenic
PRO-ANGIOGENIC
• Degradation of basement membrane and ECM to allow for cell
detachment and migration
• Cleavage of VE-cadherin cell-cell adhesion
• Release of active VEGF from ECM stores
• Cleavage of basement membrane to release bFGF and to release
and activate TGFβ
MMPsMMPs areare pro pro andand antianti--angiogenicangiogenic
ANTI-ANGIOGENIC
• Generation of antiangiogenic factors
- angiostatin from plasminogen
- endostatin, tumostatin, arrestin, and canstatin from type
XVIII and IV collagen
Tissue inhibitors of
metalloproteinases (TIMPs)
Matrix metalloproteinases (MMPs)
zinc-dependent endopeptidases
Balance between MMPs and TIMPsBalanceBalance betweenbetween MMPsMMPs andand TIMPsTIMPs
StagesStages ofof angiogenesisangiogenesis
1. increase in vessel permeability
2. loosening of pericyte contact
3. proteinase release from endothelial cells
4. digestion of basement membrane and extracellular matrix
5. migration and proliferation of endothelial cells
6. formation of vascular structures
7. fusion of new vessels
8. initiation of blood flow
inhibition of endothelial cell proliferation
inhibition of the migration of endothelial cells
9. formation of basement membrane
Angiogenesis - Endothelial Cell Migration
Endothelium
Nascent
Vascular Sprouts
VEGF
Smooth Muscle
Cells
Basement
Membrane
StagesStages ofof angiogenesisangiogenesis
1. increase in vessel permeability
2. loosening of pericyte contact
3. proteinase release from endothelial cells
4. digestion of basement membrane and extracellular matrix
5. migration and proliferation of endothelial cells
6. formation of vascular structures
7. fusion of new vessels
8. initiation of blood flow
inhibition of endothelial cell proliferation
inhibition of the migration of endothelial cells
9. formation of basement membrane
Angiogenesis Angiogenesis -- Endothelial Cell ProliferationEndothelial Cell Proliferation
Endothelium
VEGF
Sprout
ElongationSmooth Muscle
Cells
Basement
Membrane
Angiogenesis Angiogenesis -- Capillary MorphogenesisCapillary Morphogenesis
Endothelium
VEGF
New
Lumen
FormationSmooth Muscle
Cells
Basement
Membrane
StagesStages ofof angiogenesisangiogenesis
1. increase in vessel permeability
2. loosening of pericyte contact
3. proteinase release from endothelial cells
4. digestion of basement membrane and extracellular matrix
5. migration and proliferation of endothelial cells
6. formation of vascular structures
7. fusion of new vessels
8. initiation of blood flow
inhibition of endothelial cell proliferation
inhibition of the migration of endothelial cells
9. formation of basement membrane
Angiogenesis Angiogenesis -- Vascular MaturationVascular Maturation
Endothelium
VEGF
SMC, pericyte
recruitment
Smooth Muscle
Cells
Basement
Membrane
SummarySummary ofof thethe mechanismsmechanisms ofof angiogenesisangiogenesis
arterio/venous
differentiation(ephrins/Eph)
Vessel maturation
and regression
AngiogenesisAngiogenesis isis dependent on dependent on thethe balancebalance
betweenbetween propro-- andand antianti--angiogenicangiogenic mediatorsmediators
VesselVessel maintainancemaintainance versusversus vesselvessel regressionregression
Carmeliet, Nature Med. 2003
AngiogenesisAngiogenesis maymay be be impairedimpaired inin many many diseasesdiseases
CardiovascularCardiovascular
diseasesdiseases
VEGF, bFGF, EGF
tumor
Small avascular
tumorTumor secretion of angiogenic factors
is triggered by hypoxia
VEGF, bFGF, EGF
tumor
hypoxia in
the center of tumorANGIOGENIC SWITCH
Pro-angiogenic factors stimulate the migration and
proliferation of endothelial cellsRapid tumor growth and metastasis
VEGF, bFGF, EGF
Tumor
expansion
Tumor Tumor angiogenesisangiogenesis
Vessel intussusception
EPCs Lymphangiogenesis
blood vessel
tumor
EPC
tumor
blood vessel
bone
marrowtumor
lymphatic vessel
DifferentDifferent mechanismsmechanisms ofof bloodblood formationformation inin tumorstumors
Loboda et al., 2011
DifferentDifferent mechanismsmechanisms ofof bloodblood formationformation inin tumorstumors
Vasculogenic mimicry
tumor
endothelial cell
VM cell
Vessel co-option
blood vessel
tumor
Loboda et al., 2011
TheThe mechanismsmechanisms involvedinvolved inin bloodblood vesselvessel formationformation areare
evolutionaryevolutionary veryvery conservativeconservative
Schweitzer et al. Science 2005.
DissolvedDissolved T. T. rexrex bonebone yieldedyielded flexibleflexible, , branchingbranching
vesselsvessels somesome ofof whichwhich containcontain cellcell--likelike structuresstructures
PresencePresence ofof VEGFVEGF--likelike proteinsproteins inin differentdifferent animalsanimals
• In the nematode Caenorhabditis elegans four possible
homologs of PDGF/VEGF receptors (VER-1 to VER-4) and
one ligand (PVF-1) are known
• PVF-1 has the ability to bind to human receptors VEGFR-
1 and VEGFR-2 and to induce angiogenesis in two model
systems derived from vertebrates
Jorgensen & Mango, Nat Rev Gen 2002; Tarsitano et al. FASEB J 2006.
Control HUVEC HUVEC + VEGF HUVEC + PVF-1
O’Farrell, J Clin Invest 2001
DrosophilaDrosophila melanogastermelanogaster
respiratory (respiratory (trachealtracheal) system) system
- Branching tubular system of trachea delivers oxygen to
the tissues of insects.
- Its development shows parallels to the angiogenesis
- Branchless (a homolog of mammalian FGF), PVF1, PVF2, PVF3 (homologs of
mammalian VEGF/PDGF) and PVR receptor regulate the migration of early
hemocytes and are necessary for formation of tracheal system.
Tracheal tree of Drosophila embryo
DB – dorsal branch; DT – dorsal trunk;
GB – ganglionic branch; VB – visceral branch
O’Farrell, J Clin Invest 2001
DrosophilaDrosophila melanogastermelanogaster
respiratory (respiratory (trachealtracheal) system) system
- The cellular morphogenesis by which insect tracheal
cells produce fine terminal branches resembles capillary
formation of endothelial cells.
- Like capillaries, the branching of terminal trachea is
not sterotyped, but is regulated by the availability of
oxygen.
- Despite the extensive differences in their designs, the oxygen
delivery systems of insects and mammals may
have evolved from a common primitive system
present in evolutionary predecessors of
both groups.
Tracheal system in the
muscle.
(Corrosion cast)
Trachea
Take home messages
1. Endothelial cells form the inner part of blood vessels
2. Three main mechanisms of formation of blood vessels are
known
3. Numerous mediators are involved in blood vessels formation
4. Physiological angiogenesis in adults is restricted, but it is a
significant component of numerous diseases, such as cancer
or atherosclerosis
5. Angiogenesis is a multi-step process including activation of
endothelial cells, their proliferation and migration