lectureI 5th March2012 - Jagiellonian...

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.


• 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


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).


• 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


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

Angiogenesis - Endothelial Cell Migration

Endothelium

Nascent

Vascular Sprouts

VEGF

Smooth Muscle

Cells

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

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

lectureI 5th March2012 - Jagiellonian...

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