Lecture VI

Arteriogenesis

Angiopoietins, ephrinsand others

19th April 2010

EC – endothelial cellP – pericyteF – fibroblastVEGF – vascular endothelial

growth factorPDGF – platelet-derived

growth factorAng-1 – angiopoetin-1

Mechanisms of new blood vessels formation

Vasculogenesis

EC

P

EndothelialProgenitor

Cell

Capillaryblood vessel

Angiogenesis

EC

P

EC

EC

P

EC

P

Capillaryblood vessel Network of capillaries

VEGF

Arteriogenesis

EC

P

EC

SMCSMC

SMCSMC

F

F

Primaryblood vessel

Mature artery

increased blood flowVEGF + PDGFVEGF + Ang-1

Arteriogenesis- collateral circulation

This is a process in which small (normally closed) arteries open up andconnect two larger arteries or different parts of the same artery. Theycan serve as alternate routes of blood supply.

The newly formed arteries, clinically described as collaterals, develop to bypass severe arterial stenoses to connect the proximal (high-pressureregion) to the distal (low-pressure region) arterial system

Differences BetweenAngiogenesis and Arteriogenesis

AbleUnableCompensation for anoccluded artery

10- to 20-fold1.5- to 1.7-foldIncreases blood flowmaximally

Inflammation because ofincreased shear stress

Inflammation because ofischemic focal tissue damage

Cellular mechanism

Shear stressIschemiaTrigger

Pre-existing arteriolesPre-existing capillariesSource

Growth of collateral arteriesFormation of new capillaries by sprouting

Definition

ArteriogenesisAngiogenesis

Van Royen et al.. Cardiovasc Res 2001

Growth of collateral blood vessels (arteriogenesis) is potentiallyable to preserve structure and function of organs after occlusionof a major artery.

The remodeling process depends on the following conditions:

(1) existence of an arteriolar network that connects the preocclusivewith the postocclusive microcirculation;

(2) activation of the arteriolar endothelium by elevated fluid shearstress;

(3) invasion (but not incorporation) of bone marrow–derived cells(4) proliferation of endothelial and smooth muscle cells.

Arteriogenesis• refers to an increase in the diameter of existing arterialvessels

mechanical stimulation chemical stimulation

elevated blood pressure andincreased blood flow

upregulation of cytokinesand cell adhesion receptors

VEGF, MCP-1, TNF-α, bFGF, MMP, angiopoietins,

Waltenberger 2001, Circulation

Arteriogenesis in collateral development – involvementof endothelial cells, smooth muscle cells and monocytes

1. The initial triggers of arteriogenesis are physicalforces like fluid shear stress

2. Attraction and invasion of circulating blood cells –monocytes

3. Proliferation of vascular wall cells4. Remodeling processes with digestion and

rearrangement of the extracellular matrix and elasticlamina.

Arteriogenesis – steps

Arteriogenesis – steps

This series of electron microscopic figures shows subsequent steps of thecollateral vessel wall invasion by blood monocytes

Heil, Schaper Circ Res 2004

Monocytes in collateral artery growth

VSMC proliferation and remodeling

Heil, Schaper Circ Res 2004

Arteriogenesis – steps

Role of VEGFR-1 in arteriogenesis –stimulation of monocyte migration

Waltenberger 2001, Circulation

PlGF

VEGF family

Integrins E, P selectin VEGFR-1 VEGFR-2 TIE-2 TIE-1

sVEGFR NRP-1 VEGFR-3

ephrins

IgG superfamily(VCAM-1, ICAM-1)

Ang1

Ang2

Ang3

Ang4B1 A1 B2

B2 B3 B4 A2ephrin receptors

A B C D E PlGF

VEGF

?

+

--

+

Diversity of angiogenic factors/receptors

Jain and Munn Nature Med. 2000, modified

Carmeliet, 2005; Semenza 2003

Blood vessel formation – various ways

Yancopoulos, Science 2000

Receptorson endothelial

cells

Two classes of endothelial cell specific tyrosine kinase receptors

Jones N et al., Nature Cell Biol 2001

Ig-like domain

EGF-likecysteine repeats

Fibronectin type III homology domains

Tie-1 and Tie-2 receptors

1. Endothelial cell specific tyrosine kinase receptors

2. Built of extracellular and intracellular domains – different thanVEGF receptors

3. Expressed early in development, especially in endothelial cells

4. Tie-1/Tie-2 – specific markers of EC, are also present on certain hematopoietic cell types

5. Tie –2 - receptor for angiopoietinsTie –1 - ?

69% 63%

AngAng--11

AngAng--22

coiled-coil domain fibrinogen-like domain

responsible foroligolimerization pattern

mediatesreceptor phosphorylation

C

C

N

N

44%

42% 55%

57% AngAng--33

AngAng--44C

C

N

N

Angiopoietins

Knockouts of Tie-1 and Tie-2 receptors

Tie-1 -/- die between E13.5 and E18.5normal development up to E13.0; then local hemorrhage,

edema, rupture of microvessels

Tie-2 -/- die at E10.5 cardiac failure, hemorrhage, other vascular defects

embryos have low number of endothelial cells, lack of smoothmuscle cells

Ang-1 1. Ligand for Tie-2

2. Does not induce endothelial cell proliferation, but is required for their survival

3. Expressed in the myocardium and perivascular cells

4. Ang-1-/- - defects similar to Tie-2-/-

- death of embryos at E12.5, - lack of stabilization and remodelling of vessels – lack of theinteraction between endothelial and supporting cells

Ang-1 activities

• is responsible for sprouting angiogenesis (like VEGF) but Ang-1 is a weak mitogen for endothelial cells

• gene knockout studies have demonstrated that VEGF is necessary for the formation of the initial vascular plexus during embryogenesis, whereas Ang-1 is essential for subsequent remodeling, maturation and stabilization of vessels

• the mechanism of Ang-1 on migration, tube formation and survival of endothelial cells in vitro is mediated via PI3K and downstream effectors of the cascade

• overexpression of Ang-1 leads to strong vasculature, probably due to the promotion of remodeling and inhibition of pruning

Ang-1 activities

Central role of Akt kinase in angiogenesis signaling

Dimmeler & Zeiher, Circ Res 2000

Endothelial cell apoptosis and Ang-1

p85, p110 – subunits of PI3K

Overexpression of Ang-1 in theskin of transgenic mice

Increased Vascularization in MiceOverexpressing Angiopoietin-1

Suri et al, Science 1998

skin phenotypein newborn

Skin phenotype in adult Ang1 transgenic mice –big differences

Increased Vascularization in MiceOverexpressing Angiopoietin-1

Suri et al, Science 1998

Ang-1 exerts effects which areopposite to VEGF action

• in contrast to VEGF, which is one of the strongest vascular permeability factor, Ang-1 decreases vessels permeability. In this way Ang-1 can protect blood vessels from the VEGF activity and connected tissue edemas

• Ang-1 overexpression may have also an anti-inflammatory effect. VEGF stimulates endothelium to produce tissue factor and induces inter-cellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1) and E-selectin expression which leads to the adhesion of leukocytes. Ang-1 may protect from these harmful effects of VEGF.

Ang-1 has anti-permeability and anti-inflammatory functions

VEGF vascular permeability factor (VPF)

VCAM-1 ICAM-1 E-selectin

adhesion of leukocytes

Thurston et al., Nature Med. 2000

Strong VEGF expression induces massive edema –VEGF is also a vascular permeability factor

massive edema in fourorgans 36 h afterintravenous administrationof Ad–VEGF or Ad–GFP

Thurston et al., Science 1999

Comparison of untreated ear skin of wild-type, K14-VEGF,and K14-Ang1 mice

The skin of K14-VEGF mice was redder thannormal, but, unlike theskin of K14-Ang1 mice, itwas also thicker thannormal, with regions ofincreased cellularity andfoci of inflammatory cells.

Thurston et al., Science 1999

increases invessel length

increases invessel diameter

Thurston et al., Science 1999

Comparison of plasma leakage in ear skinafter treatment with inflammatory agents

mustard oil - an inflammatoryagentthat induces plasma leakageand inflammationin the skin

Evans blue dye to visualizeleakage

Thurston et al., Nature Med. 2000

Systemic Ang-1 production by adenoviral gene delivery causesresistance to vascular leakage induced by mustard oil andVEGF

Ears of mice treated withAd–Ang1 or Ad–GFP and, 3 d later, injectedintravenously with Evansblue dye to visualizeplasma leakage, followedby exposure to saline ormustard oil for 30 min.

• viral overexpression of Ang-1 increases lymphangiogenesis in mouse skin and cornea

• influence of Ang-1 on lymphangiogenesis is connected with upregulation of VEGFR-3

Thus, similarly to Ang-1/Ang-2 and VEGF cooperation in blood vessel development, crosstalk with VEGF-C and D during formation of lymph vessels can occur

Ang-1 in lymphangiogenesis

Formation of filopodia andendothelial sprouts was observedalready 2 days after adenoviraldelivery of Ang1 and VEGF-C

At 4 days, the sprouting andendothelial activation increased inthe Ang1-treated and VEGF-C ears

After 14 days, both Ang1- andVEGF-C–treated samples showedvessel network formation andstabilization of the lymphaticendothelium

Angiopoietin-1 promotes lymphaticsprouting and hyperplasia

Tammela et al.. Blood 2005

Thurston F, Cell Tissue Res 2003

Angiopoietin-21. Ligand for Tie-2 - antagonist of Ang-1 in some cells

- but in others – can phosphorylate Tie-2

2. Expressed at low level by endothelial cells, but is stronglyupregulated at sites of active vascular remodeling - ovary, tumors

3. Is upregulated at times of both vessel growth andregression – thus Ang-2 plays an active role in bloodvessel remodeling

4. Ang-2 -/- - born relatively normal, but many die at 14 day –defects in remodeling of the vessels

5. Transgenic overexpression of Ang-2 disrupts blood vesselformation

Ang-2-deficient mice are born apparently normal - the functionallyunaffected blood vascular system of Ang-2-deficient mice has onlyminor abnormalities

In contrast to the mild phenotype of Ang-2-deficient mice, micetransgenically overexpressing Ang-2have anembryonic lethalphenotypethat essentially phenocopies the Ang-1-null and Tie-2-null phenotypes

The similarity of the Ang-1 loss-of-function phenotype with the Ang-2 gain-of-function phenotype strongly supports the antagonistic conceptof Ang-1 and Ang-2 functions

Expression of Ang-1 and Ang-2 in various tissues

Angiopoietin-2

• Ang-2 is an antagonist of Tie-2 receptor!It binds to Tie-2 without inducing signal transduction inTie-2-expressing endothelial cells

• its action is dependent on the VEGF expression

high VEGF level

Ang-2 cooperates with VEGF

enhancement of angiogenesis

absence of VEGF

blood vessels remodeling

Ang-2 is upregulated in sites of vessels regression

Ang-2 and VEGF

The Tie-2 ligand Angiopoietin-2 destabilizesquiescent endothelium

HUVECs - human umbilical-vein endothelial cellsHUASMCs - human umbilical artery smooth-muscle cells

A-422885.66 - the low-molecular-weight Tie-2 inhibitor

Three dimensional co-culture model of endothelial cells and smooth-muscle cells was used. In this model, smooth-muscle cells form thecore of a spheroid that is covered by a monolayer of endothelial cells. The surface endothelial monolayer can be visualized by whole-mountCD31 or ICAM-1 immunocytochemistry

Scharpfenecker M et al. J Cell Sci 2005

Scharpfenecker, M. et al. J Cell Sci 2005

Induction of endothelial-cell detachment from endothelial-cell/smooth-muscle-cell co-culture spheroids by

pharmacological Tie-2 inhibition

Scharpfenecker, M. et al. J Cell Sci 2005

Time course of Ang-2-mediated endothelial-cell-monolayer destabilization in HUVEC/HUASMC co-culture spheroids

Scharpfenecker, M. et al. J Cell Sci 2005

Destabilization of endothelial-cell-monolayer integrity in co-culture spheroids of ECs and SMCs by exogenous Ang-2

Angiopoietin-2 displays VEGF-dependent modulation ofcapillary structure and endothelial cell survival in vivo

Lobov et al.. PNAS 2002

Ang2 induces rapidcapillary diameterincrease and EC shapechange

The pupillary membrane -uniquein vivo model system. This structure is a temporary vascular networkthat surrounds the anteriorpart of the lens in thedeveloping eye.

Angiopoietin-2 displays VEGF-dependent modulation ofcapillary structure and endothelial cell survival in vivo

Lobov et al.. PNAS 2002

Capillary regression is induced by VEGF inhibition and promoted by Ang2.

Cells with apoptotic nuclearmorphology are indicated

Early development Late development &Adult

VEGF endothelial cells Ang1 vessel maturationdifferentiation (agonist) & stabilizationproliferationtube formation

Ang2(antagonist)

vessel de-stabilization

+ VEGF No VEGF

Adult neovascularization Vessel regression

Angiopoietin-3 and angiopoietin-4

•they bind to Tie2 receptor

• in experiments with human endothelial cell lines, Ang3 was identified as an antagonist of Tie2 andAng4 was identified as an agonist of Tie2

Ang4, but not Ang3, strongly induces Tie2 and Akt phosphorylations in HUVECs

Lee et al., FASEB Journalmouse Ang3 and human Ang4 were used

Ang4, but not Ang3, strongly inducessurvival and migration in HUVECs

Lee et al., FASEB Journal

Ang3 is more potent than Ang4 in Akt phosphorylationand survival in primary cultured mouse ECs

Both Ang3 and Ang4 induce strong angiogenesis in vivo

angiogenesis in the mouse corneal micropocket assay

Lee et al., FASEB Journal

Model for the action of Ang3 and Ang4 in human Tie2 and mouse Tie2. Ang4 is a relatively potent agonist whereas Ang3 is a very weak agonist to humanTie2. In comparison, Ang3 is a relatively potent agonist whereas Ang4is a moderate agonist to mouse Tie2.

Lee et al., FASEB Journal

Integrins E, P selectin VEGFR-1 VEGFR-2 TIE-2 TIE-1

sVEGFR NRP-1 VEGFR-3

ephrinsIgG superfamily

(VCAM-1, ICAM-1)

Ang1

Ang2

Ang3

Ang4B1 A1 B2

B2 B3 B4 A2ephrin receptors

A B C D E PlGF

VEGF

?

+

--

+

Diversity of angiogenic factors/receptors

Jain and Munn Nature Med. 2000, modified

Ephrins and Eph receptors

Eph receptor tyrosine kinase family and their ligands, ephrins, are critical regulators of vascular remodelingduring embryogenesis and in tumor neovascularization

• ephrins (Eph family receptor interacting proteins)

• the Eph receptors family (Erythropoietin-producing human hepatocellular carcinoma) is the largest known family of receptor tyrosine kinases consisting of 16 members

• both Eph receptors and their ligands are divided in two subfamily – A and B

• ligands of EphrinA class are glycosylphosphatidylinisotol (GPI)-anchored peripheral membrane molecules, whereas ephrinB subfamily is formed by transmembrane proteins with cytoplasmicdomain

Ephrin receptors and ephrin ligands

Augustin & Reiss, Cell Tissue Res, 2003

Yancopoulos, Science 2000

Receptorson endothelial

cells

Eph receptors are present in high degrees during vasculogenesis andearly development of the circulatory system

• They play a role in

- distinguishing between arterial and venous endothelium,- stimulation of the production of capillary sprouts- differentiation of mesenchyme into perivascular supportcells

Role of ephrin/Eph receptorsin angiogenesis

Ephrin B2 – an early marker of arterial endothelial cells

EphB4 – marks venous endothelial cells

Ephrins and Eph receptors –markers of arterio-venous identity

EphrinB2 -/- - lethal at E10.5 Significant defects in vessels remodeling and sprouting

EphB4 -/- – also die at E10.5

Blood vessel defects in ephrinB2 null mutant embryos and yolk sacs

growth retardation in ephrinB2 null mutant embryos

the normal formation of blood islands andprimary capillary plexus in wild-type, but failure of angiogenic remodelling intolarger vessels in the mutant yolk sac.

Vasculogenesis

Lymphangiogenesis

Angiogenesis

Mesoderm formation

Hemangioblasts Blood island formation

Endothelial cells

Hematopoietic cells

Primary vascular plexus

Lymphatics

Lymphangioblasts

VeinsCapillaries

Arteries

VE-CadherinbFGF

VEGF AVEGFR-1/2/3TGFββββEphrinB2/EphB4Endoglin Notch

Ang1/2Tie2PDGF BId1/3

VEGF C/DVEGFR-3

VEGF AVEGFR-2TGFββββ

Vascular Development

Karkkarnin et al., 2002 Nature Cell Biology

Notch on Notch off

Notch is a large transmembrane receptor that is importantfor normal neurogenesis, somiteformation and lymphoid cell development. It plays a special role in blood vessel development.

VEGF-A

Notch1,4 Dll4

Hey1,2

EphB4 ephrinB2

Venous EC

Arterial EC

Arterial/Venous Specification

Cross-talk between ephrinB-EphB4 and VEGF/Ang1

VEGF Ang1

PI3K kinase

Akt

EC survival

MAPK

EC proliferation

EphrinEphrin B2B2

Arterial EC

Evidence for Importance of Notch in Regulating Vascular Formation

• Notch -/- mouse embryo: primary plexus is formedbut there is lack of large and small vessels

• CADASIL (CerebralAutosomalDominant Arteriopathy withSubcorticalInfarcts andLeukoencephalopathy), a human diseasecharacterized by early adult onset stroke anddementia, is caused by mutations inNotch3(and itsetiology has been ascribed to a vascular defect

Ephrins/Eph in tumors

• Overexpression of Eph receptors has been detected in different types of solid tumors

• First evidence come from 1987 when EphA1 was found to be more than 10-fold overexpressed in human hepatocellular carcinoma cell line

• EphA1 was found to be expressed also in liver, lung, breast andcolon carcinoma

• EphA2 is upregulated in ovarian, lung, liver, gastrointestinal, prostate cancers as well as melanomas and glioblastoma

• The expression of other members of the Eph receptors and also ephrins is increased in a number of cancers and tumor cell lines

Integrins E, P selectin VEGFR-1 VEGFR-2 TIE-2 TIE-1

sVEGFR NRP-1 VEGFR-3

ephrins

IgG superfamily(VCAM-1, ICAM-1)

Ang1

Ang2

Ang3

Ang4B1 A1 B2

B2 B3 B4 A2ephrin receptors

A B C D E PlGF

VEGF

?

+

--

+

Diversity of angiogenic factors/receptors

Jain and Munn Nature Med. 2000, modified

• VE-cadherin is a calcium-dependent cell-cell adhesionglycoprotein build of five extracellular cadherin repeats, a transmembrane region and a highly conserved cytoplasmictail

• VE-cadherin interacts withα-, β- andγ-catenin

• VE-cadherin can signal with VEGFR-2 to mediate PI3K/Akt-dependent endothelial cell survival

Role of VE-cadherin in angiogenesis

VE-cadherin and angiogenesis

Jones et al., Nature Rev 2001

VE-cadherin is indispensable for propervascular development

Gory-Fauré et al. Development 1999

the mutant embryo:growth retardation, anemia, pericardial hypertrophy andincomplete folding.

External appearance of E10.5 yolk sacs. Bloodislands of the homozygous mutants remainedisolated (B,D), as opposed to the organizedvasculature of the wild type (A,C).

Role of VE-cadherin in angiogenesis

Disruption of VE-cadherin impairs angiogenesis – knockouts die at E9.5 Deficiency of VE-cadherin blocked the capacity of endothelial cells to respondto survival signals induced by VEGF-A, by preventing formation of a complexconsisting of VE-cadherin, β-catenin, PI3-K, and VEGF receptor-2 (VEGFR-2/Flk1/KDR)

Carmeliet et al., Cell 1999

AKT

survival

VE-cadherin VEGF-A

VEGFR2

PI3-Kββββ-cat

Integrins E, P selectin VEGFR-1 VEGFR-2 TIE-2 TIE-1

sVEGFR NRP-1 VEGFR-3

ephrins

IgG superfamily(VCAM-1, ICAM-1)

Ang1

Ang2

Ang3

Ang4B1 A1 B2

B2 B3 B4 A2ephrin receptors

A B C D E PlGF

VEGF

?

+

--

+

Diversity of angiogenic factors/receptors

Jain and Munn Nature Med. 2000, modified

Vascular integrinsA large number of vascular cell surface glycoproteins thatfunction as cell adhesion receptors to extracellular matrix

and sometimes in cell-cell adhesion

Stupack and Cheresh, Science STKE, 2002

Integrins

Pro- and anti-angiogenic role ofααααvββββ3 integrin

• Vitaxin is in Phase II trial for colorectal cancer

• a humanized version of the antiangiogenic antibody LM609 directedagainst a conformational epitope of theαvβ3 integrin complex

Vitaxin

• a promising angiogenesis inhibitor used in the treatmentof someforms of cancer

Abegrin

• Abegrin is a monoclonal antibody to human integrinαVβ3, a celladhesion molecule highly expressed on actively angiogenic endotheliumand glioblastoma multiforme tumor cells

LM-609; Vitaxin 2αvβ3 antibodies

Abegrin™ inhibits human melanoma tumor cell growthin vivo. Mice harboring either M21 (A) or A375 (B) melanoma xenografts weretreated with 10 mg/kg Abegrin™ delivered thrice weekly starting on day 1 following tumor implantation.

M21 A375

Mulgrev, Mol Cancer Ther. 2006

Summary of the mechanisms of angiogenesis

arterio/venous

Differentiation(ephrins/Eph)

Take home messages

• Arteriogenesis is the third way of blood vessels formationstimulated by elevated shear stress

•Ang1 and Ang2 act via Tie-2 receptor, but Ang-1 is anagonist whereas Ang-2 antagonist

• Ang-1 protects from increased permeability

• ephrin/Eph receptors are markers of arterio-venousidentity

• adhesion molecules (VE-cadherin, integrins) playimportant role in angiogenesis