Angiogenesis

(1)

Gerald Prager, M.D.

Medical University of Vienna

Department of Medicine I

[email protected]

Angiogenesis - Objectives

• Explain differences between vasculogenesis and

angiogenesis and collateral vessel growth

• Describe the most relevant angiogenic molecules

(cells, growth factors, matrix proteins, adhesion

molecules)

• Explain the role of angiogenesis in tumorigenesis

and wound healing

• Describe the rational behind pro-, and angiogenic

therapies

(2)

Case I

• 35a female patient, upon self examination she discovered a

tumor in the left breast

• Radiologist evaluate this as a benign tumor, because no

blood vessels were revealed in duplex-sonography

• At a 6 month setup vascularization was detected

• Tumor was resected and it turned out to be malign; ER

negativ; no lymph node, no adjuvant therapy

• After five years the patient suffered massiv pain from the

back bone, bone metastases were detected.

• The patient did not respond to any radiatio- or

chemo-therapy.

Æ

Therefore, an experimental anti-angiogenetic

therapy was started.

(3)

Angiogenesis Skin Model

5d

10d

15d

20d

Angiogenesis versus Vasculogenesis

pO2

VEGF

(4)

Vasculogenesis, Angiogenesis,

Arteriogenesis

• Vasculogenesis

new vessel formation

• Hemangioblasts

• VEGF family, Ang-1,2

• Angiogenesis

Outgrowth from pre-exisiting vessels

• Endothelial cells

• FGF-1,2,4,5; VEGF-1,2,3

• Arteriogenesis

Maturation

•ECs, SMCs, Monocytes

•PDGF, FGFs, Ang-1,2; MCP-1

•Arteriole formation

Embryonal

Adult

Moore MA. J Clin Invest 2002;109:313-315.

(5)

Postnatal angiogenesis and vasculargenesis

Angiogenesis

• cancer

• psoriasis

• arthritis

• blindness

• obesity

• asthma

• atherosclerosis

• infectious disease

• heart ischemia

• brain ischemia

• neurodegeneration

• hypertension

• pre-eclampsia

• respiratory distress

• osteoporosis

Pathological angiogenesis

insufficient or abnormal

vascular remodelling

Physiological angiogenesis

• Wound healing

• Ovulation

• Menstruation

• Implantation

(6)

Blood Vessel Formation

Vasculogenesis

Hemangioblasts

Angiogenesis

Endothelial sprouts

_{(Hemangiomas)}

VEGF

PDGF

Strom

a

Angiopoietin I

TIE 2

Angiopoietin II

VEGF

Mature blood vessel

TF

SMC

EC

Arteriogenesis

Primitive endothelial tubes

pericytes

SDF-1a

Arterial versus venous

• Thickness of SMC wall

• SMC have distinct origin (neural crest

– thoracic vessels, epicardium –

coronary artery, others from

mesenchym)

• Notch pathway (Notch-1, Notch-3 and

Notch-4: receptors) and (Delta-like-4,

Jagged-1 and Jagged-2) is important

for arterial differentiation by repressing

venous differentiation. VEGF and

Hedgehog act upstream and Gridlock

downstream of the Notch pathway.

• Neuropilin-1 (a VEGF receptor) also

(7)

Tumor angiogenesis

Stetler-Stevenson JCI 1999; 103: 1237-1241

I. Growth Factors

• vascular endothelial growth factor (VEGF)

• fibroblast growth factor (FGF-2)

• angiopoetin (Ang1, Ang2)

• platelet derived growth factor (PDGF)

• hypoxia inducible factor (HIF-1)

• transforming growth factor (TGF-b)

• tumor necrosis factor (TNFa)

• CXCR-1, CXCR-3

II. Hypoxia

III. Inflammatory Cytokines

(8)

Vascular Endothelial Growth Factors

VEGF-A:

Smooth muscle cells, Keratinocytes, Epithelial cells

Tumor Cells, Fibroblasts, Makrophages;

Vascular permeability, Endothelial cell proliferation,

Angiogenesis

VEGF-B:

Striated muscle cells (heart and muscle);

Angiogenesis

VEGF-C:

Heart, Placenta, Tumor cells, small intestine;

Lymphangiogenesis

VEGF-D:

Lung, Skin;

Mitogen for endothelial and fibroblasts

PlGF-1, 2:

Placenta;

synergistic with VEGF-A

VEGF and VEGF Receptors

PLGF1, 2

VEGF-A

VEGF-B

VEGF-C

VEGF-D

VEGFR-3

(flt-4)

VEGFR-1

(flt-1)

VEGFR-2

(flk-1)

Migration

Migration, Proliferation, Permeability

Proliferation

Endothelial Cell Lymphatic-Endo thelial Cell

RTK

PKC

sVEGFR-1

SMC, Keratinocytes, Epithelial, Macrophages

Striated MC

Placenta

Tumor cells

Lung, Skin

small intestine

Splice variants:

Secreted: +/- Heparin binding

Membrane/Matrix bound

(9)

Binding of VEGF to its Receptors

VEGF

VEGFR

PLC IP₃ Ca++ PKCε

α β

Integrins DEP-1 fyn FAK FAK PI3, 4P Src Src RAF MEK-1/2 ERK-1/2 EGR EGR--11 T

Tff, flt, uPA / uPAR, flt, uPA / uPAR others others Ras-GTP 14-3-3 PDK2 Akt Cdc42/RAC JAK JAK STAT PAK

+

-TAB XIAP TAK IKK2 NFκB NF NFκκBB Inflammatory mediators Inflammatory mediators MEK 3/6 PI3 PI3 K K u-PAR GP130 IκB-P

Antiapoptotic

Dia ROK MLCK Lamellipodia Migration Tube-Formation Tyrosine substrates PI4P SOS SOS Calcineurin NFAT cas paxSrc Src P 38 u-PAR

?

_{LRP M6P-R} PAI-1 RTK

-PLCγ

Monocyte

binding

cPL cPL A A₂2 lipoxygenase PPAR PPARγγ Gαs Lox-1 Gαq Microtubuli

vvo

?

PKA

cAMP

-CREB

CREB

CBP/p3oo

Cell cycle

TFs, Myc TFs, Myc Cycl D Cycl D CDK CDK 4 4 ARF ARF MDM MDM 2 2 p53 p53 Rb Rb Rb Rb--PP E2F E2F

-CDI

CDI--KipKip

+

-HOMO E-3-L E-2-T x E-2-T TSG101

-Neutrophil

binding

LIMR Stress-fibres TGFβ others others NIK MEKK 1 MEK-4/7 JNK TNF-R

-TRAF TRAF Nab Nab--22

-12 HETE VEGF FGF EGF Pgen Tumor cell Tumor cell Protease X CYR61 TSP like

?

_Angiostatin Angiostatic effects

Ox PL

(10)

PLC IP₃ Ca++ PKCε

α β

Integrins DEP-1 fyn FAK PI3, 4P Src Src RAF MEK-1/2 ERK-1/2 EGR EGR--11 T

Tff, flt, uPA / uPAR, flt, uPA / uPAR others others Ras-GTP 14-3-3 PDK2 Akt Cdc42/RAC JAK STAT PAK

+

-TAB XIAP TAK IKK2 NFκB NF NFκκBB Inflammatory mediators Inflammatory mediators MEK 3/6 PI3 PI3 K K u-PAR GP130 IκB-P

Antiapoptotic

Dia ROK MLCK Lamellipodia Migration Tube-Formation Tyrosine substrates PI4P SOS SOS Calcineurin NFAT cas pax Src P 38 u-PAR

?

_{LRP M6P-R} PAI-1 RTK

-PLCγ

Monocyte

binding

cPL cPL A A₂2 lipoxygenase PPAR PPARγγ Gαs Lox-1 Gαq Microtubuli

vvo

?

PKA

cAMP

-CREB

CREB

CBP/p3oo

Cell cycle

TFs, Myc TFs, Myc Cycl D Cycl D CDK CDK 4 4 ARF ARF MDM MDM 2 2 p53 p53 Rb Rb Rb Rb--PP E2F E2F

-CDI

CDI--KipKip

+

-HOMO E-3-L E-2-T x E-2-T TSG101

Neutrophil

binding

LIMR Stress-fibres TGFβ others others NIK MEKK 1 MEK-4/7 JNK TNF-R

-TRAF Nab Nab--22

-12 HETE VEGF FGF EGF Pgen Tumor cell Tumor cell Protease X CYR61 TSP like

?

Angiostatin

Ox PL

Gene Defective Mice

VEGF A -/- :

• Delayed differentiation of ECs

• Reduced sprouting

• Altered lumen formation

LETHAL

• Embryonic development normal

• Altered wound healing

• Normal Fertility

• EC development disrupted

• Normal EC Development

• Channel formation disturbed

• Increased EC proliferation

VEGF A +/- :

PlGF -/- :

VEGFR-1 -/- :

VEGFR-2 -/- :

LETHAL

(11)

Regulation of VEGF Expression

IL-1

TNF-α

PDGF

FGF-4

IGF-1

HGF

KGF

TGF-

β

Hypoxia

→

HIF-1

VEGF

↑

VEGF

Tumor supressor genes:

vHL

p53

NO

Growth factors:

Inflammatory Cytokines:

NOS

↑

↓

die aktivierte Endothelzelle

Die Angiogenese ist ein entscheidener Mechanismus der Pathogenese maligner Erkrankungen und ist

in den letzten Jahren als therpeutisches Ziel ins Zentrum des Interesses gerückt. Ein besseres

Verständnis der zugrundeliegenden Mechanismen ist Grundvorraussetzung für effektive

therapeutische Ansätze.

Schritte der aktivierten Endothelzelle im Rahmen der Angiogenese:

1. Lösung der Zell-Zell-, sowie der

Zell-Matrix-Kontakte

2. Transmigration durch die Basalmembran in

umliegende Matrix

3. Zell-Proliferation

4. Zell-Polarisation zur Formierung

Kapillar-ähnlichen Strukturen

5. Zellüberleben: Während all diesen Schritten sind

Endothelzellen pro-apoptotischen Einflüssen

ausgesetzt. Zellüberleben stellt daher eine

Grundvorraussetzung dar.

uPAR Matrix degradation Specific matrix Laminin uPA αvβ3/5 VEGF Ang 2 Migration Tumor FN

Proliferation Tube formation

bFGF ERK 1-2

DEP-1 PAI-1 uPAR downregulation

(12)

Effects of VEGF on ECs

Zhhh..

MATRIX

Endothelial Cell

Blood

Stream

Blood

Stream

(13)

MATRIX

Endothelial Cell

Blood

Stream

V

E

G

F

MATRIX

V

E

G

F

Endothelial Cell

Blood

Stream

(14)

Adhesion

Proteolysis

Zhhh..

Endothelial Cell

Blood

Stream

Adhesion

Proteolysis

Zhhh..

Endothelial Cell

Blood

Stream

(15)

Steps of angiogenesis

1. EC proliferation

2. EC migration / invasion

3. EC survival

4. Capillary-like tube formation

5. Vessel maturation (pericytes)

(16)

Provided as an educational resource. Do not copy or distribute.

Angiopoietins Bind With Tie2 Receptor

Tie2 is a tyrosine kinase

receptor that can be found in

vascular endothelial cells

Ligand binding of angiopoietin-1

(Ang1) promotes Tie2 receptor

dimerization

Ang2 exerts antagonistic

functions on Ang1/Tie2 signaling

Thomas M, Augustin HG. Angiogenesis. 2009;12:125–137.

Tie2 Tie2 Ang1 Ang1 Ang2 Ang2

Pericytes

Endothelial

Cells

Provided as an educational resource. Do not copy or distribute.

(17)

Angiopoetin-1, Tie2 receptor

• Stabilize vessels

• Inhibits endothelial permeability

• Impairs vessel maturation and stabilization

Angiopoetin-2, Tie2 receptor

PDGF-BB

• Recruits smooth muscle cells

FGF-2

• Mediates EC migration, proliferation

TGF-beta

(18)

II. Hypoxia

• Hypoxia activates hypoxia-inducible transcription factor

(HIFs)

• HIFs induce expression of VEGF, NOS, PDGF, Ang2

+ myocardial infarction, stroke

- blindness in premature newborns, diabetic

III. Inflammation

• Monocytes, platelets, macrophages, other

leukocytes:

• Release of VEGF, Ang1, FGF2, TGF-b,

PDGF, TNFa

(19)

Mature blood vessel

TF

EC

Shearstress

Inflammation

Blood Vessel Formation

Monocyte

TNF

ICAM-1 MCP-1

SMC

Arteries/Arteriols

MCP-1

Molecular properties of

angiogenic endothelial

cells

(20)

Molecular properties of angiogenic endothelial cells

Tumor angiogenesis

Tumor

uPAR

Matrix degradation

Specific matrix

Laminin

uPA

α

v

β

3/5

VEGF

Ang 2

Migration

Non specific matrix

FN

Proliferation Tube formation

bFGF

ERK 1-2

DEP-1

_PAI-1

uPAR

downregulation

(21)

endothelial

cell response

A. Proteolytic enzymes

¾

urokinase/plasminogen system

¾

Matrix metalloproteinases (MMPs)

B. Adhesion molecules

¾

integrin adhesion receptors (alphaVbeta3)

0 0,5 1 1,5 2 2,5 3 5min _60min control VEGF 15m in 30m in 120m in D-dimer m g/ l

D-dimer concentrations in supernatants

D-dimer

control

VEGF

VEGF, 60min

10µM 10µM 10µM

Prager GW, et al. Blood (2004 ); 103(3): 955-962

A. Proteolytic System

VEGF induces an increase in D-Dimer

concentrations in supernatants

(22)

Ternary complex (uPAR – uPA – serpin)

binds to LDL-R like molecules:

LDL-R-family

uPA-Receptor

PAI

uPA

pro-uPA

PAI-1: plasminogen activator inhibitor-1

uPA: urokinase

LDL-R: low density lipoprotein-receptor

control

VEGF

165

(50ng/ml, 2h)

10 μm

Redistribution of urokinase receptor (uPAR)

upon VEGF stimulation

uPAR

phospho-FAK

(23)

Internalization of the tetrameric complex

and recycling of uPAR

uPAR

PAI

uPA

RAP

pro uPA

LDL-R-family

Matrigel plugs after 1 week

in vivo assay is used to test relevance of in vitro

findings

+ VEGF and bFGF

+VEGF

(24)

uPAR redistribution supports VEGF-induced

endothelial cell migration in vivo

control VEGF RAP+VEGF

M M M M M

wild type

uPAR -/-

M

uPAR

-/-wild type

RAP+ VEGF 0 20 40 60 80 100 120 140

control RAP VEGF * 0 20 40 60 80 100 120 140

control RAP VEGF RAP+ VEGF ** inv aded cells p er u nit **

in vivo

matrigel plug angiogenesis assay

inv aded cells p er u nit *

total cell number

endothelial cells

p<0.05

p<0.005

**

*

Proteolytic cleavage products

Proteolysis generates also cleavage products

that regulates angiogenesis

(25)

• Angiostatin

is produced by hydrolysis of

plasminogen either by MMP-3, MMP-7,

MMP-9 or uPA, whereby they contain 3-5

kringle domains (K1-3, K1-4, and K1-5).

(26)

18 alpha chains

8 beta chains

B: Integrin Adhesion Receptors

integrin

talin

(27)

(28)

Integrin activation

(Inside out Signaling)

Src Family Kinases (?Fyn)

ADAP

PLC

γ

Ca

2+

_DAG

PKC

Talin

α

IIb

β

3 activation

PI3-Kinase

?

integrin

talin

(29)

Platelets in Initiation of Thrombus

Formation

Von Willebrand factor

GP IIb/IIIa complex

Platelet

Fibrinogen

ADAMTS13

Expression an der

Oberfläche von

aktivierten

Thrombocyten:

Tf3=PS; Tf1=GfV;

GfVIII +vWF; TF auf

zirkulierenden

Mikropartikeln

integrin outside-in signaling is mediated

via CD98hc

integrin

FAK

_src

p130cas

PI3kinase

Rho-GTPases

cell survival

cell migration

cell proliferation

α

β

matrix

CD98hc

(30)

During endothelial cell migration integrins

have to release at the trailing end to be

redistributed to the leading edge, where

they bind to the extracellular matrix

Migration

control

VEGF

+ RAP

-

blue: beta-1

-

green: beta-3

-

red: uPAR

5µm 5µm 5µm

2. Integrin distribution

(31)

1.Angiogenesis in wound healing

Keratinocyte

Platelets

Stro

m

a cells

Monocyte

EC

VEGF

uPA

+

TGF

β

_MCP-1

HGF

TGF

β

VEGF

_PDGF

TGF

β

Thrombin

Fibrin

uPA

PAI-1

flk1

Ang 1

TNF

IL-1

Matrix

Collagen

GAG

FGF

Integrin shift

+

+/-+

+

TGF

β

FGF

VEGF

FN

Pericytes

• Derive from perivascular progenitor cells

(c-Kit+, Sca-1+, VEGFR-1+) and are

mobilzed upon PDGF-BB stimulation.

• Integrin Alpha-4 / VCAM interaction

(32)

(33)

(34)

kindly provided by Hoffmann-La Roche Ltd

VEGF

HGF

FGFs

PDGF

PlGF

TGF-β

(35)

Chaotic and mosaic vessels in

tumours

(36)

(37)

Anti-VEGF strategies

• Bevacizumab (AVASTIN): only effective

in combination with chemotherapy (colon,

breast, kidney, lung).

Immunofluorescence labeling of malignant

keratinocytes and vessels 2 wk after

implantation

Invasive behavior of malignant mouse

keratinocytes (PDVA cells), 2 wk after

implantation.

Khalid Bajouet al., JCB 2001

(38)

-/-downregulation of PAI-1

loss of PAI-1 suppresses pathological angiogenesis in

tumors, ocular and other disorders, while adenoviral

PAI-1 gene transfer reverted this phenotype

targeting MMPs

Cancer growth and angioenesis were also impaired in mice

lacking components of the MMP system (i.e., MMP-2

and MMP-9, while overexpression of MT-MMP-1

produced highly vascular tumors)

MMP-system

• Pro-MMP-2 binds to MT1-MMP

to become activated

• MT1-MMP is associated with

integrins (alphaVbeta3)

• TIMP-2 inhibits MMP-2

• Active MMP-2 cleaves laminin

collagen IV

(39)

Antiangiogenic therapeutic strategies

Substance/Approach

Comment

Syn

thetic/sem

i

syn

th

etic inh

ibitor

Carboxiamidotriazole (NCI)

CM101

Ca channel blocker, phase I Analog of group B streptococcus toxin (polysaccharide) binds to tumor endothelium, induces inflammation

Marimastat (British Biotech)

Metalloproteinase inhibitor, inhibits endothelial and tumor cell invasion, phase II

(pancreatic, lung, brain)

Pentosan polysulfats

Inhibits heparin-binding growth factors, phase I

TNP470 (Takeda/Abbott)

Analog of antibiotic fumagillin, inhibits endothelial cell migration and proliferatin

phase III (breast, Kaposi´s sarcoma, cervical)

Thalidomide (Grünewald)

Polycyclic teratogen, antiangiogenic mechanism unknown, phase II (brain, breast,