Angiogenesis

Proangiogenic - Angiogenic growth factors

VEGF, FGF, TNF, HGF, IGF,TGF, Proliferin

Anti angiogenetic - Thrombospondin -1, 2

Angiostatin

Endostatin

Platelet factor 4

AngiogenesisLocal degradation of basement membrane

Directional migration

invasion

Endothelial cell proliferation

Capillary tube morphogenesis

Coalescence of capillaries

Vascular pruning

What Is Metastasis?What Is Metastasis?The ability of cancer cells to:The ability of cancer cells to:1. Penetrate into 1. Penetrate into

Lymphatic.Lymphatic. Blood vessels.Blood vessels.

2. Circulate through bloodstream.2. Circulate through bloodstream.4. Invade and grow in normal tissues elsewhere. 4. Invade and grow in normal tissues elsewhere.

It is the ability to spread to other tissues andIt is the ability to spread to other tissues andorgans that makes cancer a potentially lifeorgans that makes cancer a potentially lifethreatening disease. threatening disease. So what makes metastasis possible for aSo what makes metastasis possible for aCancerous tumor?Cancerous tumor?

Blood vesselBlood vessel

1. Cancer cells invade surounding Cancer cells invade surounding tissues and vesselstissues and vessels

2. Cancer cells are transported by the circulatory system to distant sites.

3. Cancer cells reinvade and grow at new location

Metastasis Requires AngiogenesisMetastasis Requires Angiogenesis

Growth of a new network of blood vessels

What Is Tumor Angiogenesis?What Is Tumor Angiogenesis?Tumor angiogenesisTumor angiogenesisProliferation of a network ofProliferation of a network ofblood vessels that penetratesblood vessels that penetratesinto cancerous growths.into cancerous growths.FunctionFunctionSupplying nutrients and oxygenSupplying nutrients and oxygenand removing waste products.and removing waste products.MechanismMechanism Cance cells releas moleculesCance cells releas moleculesthat send signals to surroundingthat send signals to surroundingnormal host tissue.normal host tissue.This signaling activates certainThis signaling activates certaingenes in the host tissue that, ingenes in the host tissue that, inturn, make turn, make proteins to encourageproteins to encouragegrowth of new blood vessels. growth of new blood vessels.

Normal Angiogenesis: vasculogenesisNormal Angiogenesis: vasculogenesis

Normal Angiogenesis in AdultsNormal Angiogenesis in Adults

New blood vessels form in the lining of the uterus during the menstrual cycle.

Repair or regeneration of tissue during wound healing.

Angiogenesis and Vascular Endothelial CellsAngiogenesis and Vascular Endothelial Cells

The walls of blood vessels are The walls of blood vessels are formed byformed by vascular endothelial vascular endothelial cellscells..

These cells rarely divide, doing These cells rarely divide, doing so only about once every 3 years so only about once every 3 years on average. on average.

When the situation requires it, When the situation requires it, angiogenesis can stimulate them angiogenesis can stimulate them to divide. to divide.

Angiogenesis andAngiogenesis and Regulatory ProteinsRegulatory Proteins

No angiogenesis

Frequent celldivision

High inhibitorsHigh inhibitorsLow activatorsLow activators

Low inhibitorsLow inhibitorsHigh activatorsHigh activators

=Rare cell

division

=

Angiogenesis

Angiogenesis and Cancer

The dilatation theoryBefore the 1960s

Angiogenesis theoryAngiogenesis theory

Without Angiogenesis, Tumor Growth Stops

•With Angiogenesis, Tumor Growth Proceeds

•In another experiment designed to find out whether cancer growth can

continue when angiogenesis occurs, researchers compared the behavior of

cancer cells in two regions of the same organ. Both locations in the eye had

nutrients available, but only one could support angiogenesis. Scientists found

that the same starting injection of cancer cells grew to 1-2mm in

diameter and then stopped in the region without nearby blood vessels,

but grew well beyond 2 mm when placed in the area where angiogenesis

was possible. With angiogenesis, tumor growth continued

•What Prompts Angiogenesis?•In an experiment designed to find

out whether molecules from the cancer cells or from the

surrounding host tissues are responsible for starting

angiogenesis, scientists implanted cancer cells in a chamber

bounded by a membrane with pores too small for the cells to

exit. Under these conditions, angiogenesis still began in the

region surrounding the implant. Small activator molecules

produced by the cancer cells must have passed out of the chamber and signaled angiogenesis in the

surrounding tissue

•Activators of Angiogenesis•Once researchers knew that cancer

cells can release molecules to activate the process of angiogenesis, the

challenge became to find and study these angiogenesis-stimulating

molecules in animal and human tumors.

•From such studies more than a dozen different proteins, as well as several

smaller molecules, have been identified as “angiogenic,” meaning that they are released by tumors as

signals for angiogenesis. Among these molecules, two proteins appear to be

the most important for sustaining tumor growth: vascular endothelial

growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF

and bFGF are produced by many kinds of cancer cells and by certain

types of normal cells, too.

•The Angiogenesis Signaling Cascade

•VEGF and bFGF are first synthesized inside tumor cells and then secreted into the surrounding

tissue. When they encounter endothelial cells, they bind to

specific proteins, called receptors, sitting on the outer surface of the cells. The binding of either VEGF

or bFGF to its appropriate receptor activates a series of relay

proteins that transmits a signal into the nucleus of the endothelial

cells. The nuclear signal ultimately prompts a group of genes to make

products needed for new endothelial cell growth.

•Endothelial Cell Activation•The activation of endothelial cells by

VEGF or bFGF sets in motion a series of steps toward the creation of new

blood vessels. First, the activated endothelial cells produce matrix

metalloproteinases (MMPs), a special class of degradative enzymes. These enzymes are then released from the endothelial cells into the surrounding

tissue. The MMPs break down the extracellular matrix—support material that fills the spaces between cells and

is made of proteins and polysaccharides. Breakdown of this

matrix permits the migration of endothelial cells. As they migrate into

the surrounding tissues, activated endothelial cells begin to divide. Soon

they organize into hollow tubes that evolve gradually into a mature network

of blood vessels

•Inhibitors of Angiogenesis•Although many tumors produce angiogenic

molecules such as VEGF and bFGF, their presence is not enough to begin blood

vessel growth. For angiogenesis to begin, these activator molecules must overcome a

variety of angiogenesis inhibitors that normally restrain blood vessel growth.

•Almost a dozen naturally occurring proteins can inhibit angiogenesis. Among this group

of molecules, proteins called angiostatin, endostatin, and thrombospondin appear to

be especially important. A finely tuned balance, between the concentration of

angiogenesis inhibitors and of activators such as VEGF and bFGF, determines

whether a tumor can induce the growth of new blood vessels. To trigger angiogenesis,

the production of activators must increase as the production of inhibitors decreases

•Angiogenesis Inhibitors and Metastasis•The discovery that angiogenesis inhibitors

such as endostatin can restrain the growth of primary tumors raises the possibility that

such inhibitors might also be able to slow tumor metastasis.

•In one striking study, researchers injected several kinds of mouse cancer cells

beneath the animals' skin and allowed the cells to grow for about two weeks. The

primary tumors were then removed, and the animals checked for several weeks.

Typically, mice developed about 50 visible tumors from individual cancer cells that had

spread to the lungs prior to removal of the primary tumor. But mice treated with

angiostatin developed an average of only 2-3 tumors in their lungs. Inhibition of

angiogenesis by angiostatin had reduced the rate of spread (metastasis) by about 20-

fold

•Angiogenesis and Tumor Dormancy•It has been known for many years that

cancer cells originating in a primary tumor can spread to another organ

and form tiny, microscopic tumor masses (metastases) that can remain

dormant for years. A likely explanation for this tumor dormancy is that no

angiogenesis occurred, so the small tumor lacked the new blood vessels

needed for continued growth.•One possible reason for tumor

dormancy may be that some primary tumors secrete the inhibitor angiostatin

into the bloodstream, which then circulates throughout the body and

inhibits blood vessel growth at other sites. This could prevent microscopic metastases from growing into visible

tumors

•Cancer in Angiogenesis-Deficient Mice •Additional support for the idea that interfering with

the process of angiogenesis can restrain tumor growth has come from genetic studies of mice.

Scientists have recently created strains of mice that lack two genes, called Id1 and Id3, whose absence hinders angiogenesis. When mouse breast cancer cells are injected into such angiogenesis-deficient

mutant mice, there is a small period of tumor growth, but the tumors regress completely after a few weeks, and the mice remain healthy with no

signs of cancer. In contrast, normal mice injected with the same breast cancer cells die of cancer

within a few weeks.•When lung cancer cells are injected into the same

strain of angiogenesis-deficient mutant mice, the results are slightly different. The lung cancer cells

do develop into tumors in the mutant, but the tumors grow more slowly than in normal mice and fail to spread (metastasize) to other organs. As a

result, the mutant mice live much longer than normal mice injected with the same kinds of lung

cancer cells.

•Angiogenesis Inhibitors in the Treatment of Human Cancer

•Researchers are now asking if inhibiting angiogenesis can slow down or prevent the growth and spread of cancer cells in humans.

•To answer this question, almost two dozen angiogenesis inhibitors are currently being tested in cancer patients. The inhibitors being tested fall into several different categories, depending on their mechanism of action. Some inhibit endothelial cells directly, while others inhibit the angiogenesis signaling cascade or block the ability of endothelial cells to break down the extracellular matrix

•Drugs That Inhibit Angiogenesis Directly

•One class of angiogenesis inhibitors being tested in cancer

patients are molecules that directly inhibit the growth of endothelial

cells. Included in this category is endostatin, the naturally occurring

protein known to inhibit tumor growth in animals. Another drug,

combretastatin A4, causes growing endothelial cells to

commit suicide (apoptosis). Other drugs, which interact with a

molecule called integrin, also can promote the destruction of

proliferating endothelial cells

•Old Drug With a New Use

•Another interesting drug is thalidomide, a sedative used in the 1950s that was subsequently taken off the market because it caused birth defects when taken by pregnant women. Although this drug clearly would not be suitable for pregnant women, its ability to prevent endothelial cells from forming new blood vessels might make it useful in treating non-pregnant cancer patients.

Drugs That Block the Angiogenesis Signaling CascadeA second group of angiogenesis inhibitors being tested in human clinical trials are molecules that interfere with steps in the angiogenesis signaling cascade. Included in this category are anti-VEGF antibodies that block the VEGF receptor from binding growth factor. Another agent, interferon-alpha, is a naturally occurring protein that inhibits the production of bFGF and VEGF, preventing these growth factors from starting the signaling cascade.•Also, several synthetic drugs capable of interfering with endothelial cell receptors are being tested in cancer patients.

•On to Clinical Trials•Researchers have answered many

questions about angiogenesis, but many questions still remain. Scientists

do not know whether using angiogenesis inhibitors to treat cancer will trigger unknown side effects, how

long treatment will need to last, or whether tumor cells will find alternative

routes to vascularization. To answer such questions, human clinical trials

are currently under way.•For an updated list of ongoing and

currently planned clinical trials involving angiogenesis inhibitors,

including phone numbers for obtaining additional information, refer to the

National Cancer Institute CancerTrial ™ Web site, which has a section

devoted to Angiogenesis Inhibitors in Clinical Trials.

Tumorgrowth

Hypoxia

Growth factors

Vessels sprouting

Tumor growth

Tumor angiogenesisTumor angiogenesis

EXCESSIVEEXCESSIVE

UlcersUlcers

StrokeStroke

BlindnessBlindness

RheumatoidRheumatoidArthritisArthritis

Heart DiseaseHeart Disease SclerodermaScleroderma

InfertilityInfertility

PsoriasisPsoriasis

AIDSAIDScomplicationscomplications

CancerCancer

INSUFFICIENTINSUFFICIENT

TumorangiogenesisTumorangiogenesis

Stimulators vs. Inhibitors

„ANGIOGENIC SWITCH“

Prevascular phase Vascular phase

Proliferation - Progression - Metastases - SurvivalProliferation - Progression - Metastases - Survival

Tumor

Extra cellular matrix

Lytic enzymes

Mast cells

Recruitmentactivation

Macrophages

Angiogenic factors

Blood

vessel

Hypoxia in AngiogenesisHypoxia in Angiogenesis

Carmeliet, Nature, 2000Carmeliet, Nature, 2000

Vascular endothelial growth Vascular endothelial growth factorfactor

•Four isoforms: 121, 165, 189, 206

VEGF 121: diffusible

VEGF 165: cell surface•Two receptors: Flt-1, KDR•Endothelial cell proliferation and migration•Extracellular matrix degeneration•Vascular permeability (VPF)

Vascular MorphogenesisVascular MorphogenesisLigands and EC selective RTKsLigands and EC selective RTKs

Vascular Endothelial Growth FactorVEGF-R2 (Flk 1/KDR): angioblast/EC proliferation VEGF-R1 (Flt 1): capillary tube formation

Angiopoietin-1 / Angiopoietin-2 (-)Tie2: vessel maturation, periendothelial recruitmentTie1: intercapillary hemodynamic balance

Distinct phenotype – complimentary function

Pathological Vascular GrowthPathological Vascular Growth

Carmeliet, Nat Med, 2000Carmeliet, Nat Med, 2000

Cascade of Vessel FormationCascade of Vessel Formation

Vasodilatation Vascular permeability Extravasation of plasma proteins EC detachement and migration EC proliferation and sprouting Lumen formation Endothelial survival and differentiation Remodeling of endothelial network

Cascade of Vessels FormationCascade of Vessels FormationANGIOPLASTANGIOPLAST

Regression Regression

Extracellular matrixExtracellular matrix

•Physical barrier•Production of angiogenic factors•Reservoir of inactive growth factos (bFGF)•Macrophages: secrete GFs•Mediation of EC binding (integrins)

EC migration, signalling, apoptosis

VEGF/bFGF - MMP – matrix degradation

Interactions in Interactions in TumorangiogenesisTumorangiogenesisJones, BJUI, 1999Jones, BJUI, 1999

Cancer Cells & Tumor VesselsCancer Cells & Tumor Vessels

• Cancer cells occupy 4% of vascular surface

• Tumor intra-vasation in 2 days

• Shedding of 106 cells / day / gram

Angiogenesis in Bladder cancerAngiogenesis in Bladder cancer

•MVD = prognostic indicator in TCC

•MVD + p53 = aggressive subset identified

•bFGF higher in metastatic pts.

•VEGF, TGF- higher in TCC

•Urinary VEGF, aFGF higher in TCC pts.

•VEGF mRNA ~ recurrence + progression

TumorangiogenesisTumorangiogenesisand LN-Metastasesand LN-Metastases

T2-4 bladder cancer, n=41Cx, pelvic LN dissectionMVD (FVIII-RA), 200x

neg. LN (n=27): 56.2 MV (SD 29.5)pos. LN (n=14): 138.1 MV (SD 37.9)

p<0.0001

Angiogenesis in Prostate cancerAngiogenesis in Prostate cancer

•MVD higher in PCa•MVD ~ stage (superior to grade, preop. PSA)•MVD predictor for metastasis after Px•TURP-MVD ~ failure of radiotherapy•Biopsy-MVD (+ GS, PSA): extraprostatic ext.•Urine-bFGF: BPH > PCa > control•Serum-bFGF: PCa > BPH•VEGF? :•PSA = antiangiogenic

Microscopic Tumor ExtensionMicroscopic Tumor Extension

Capsular penetration

Seminal vesicle extension

Pelvic lymph node metastases

Pathologic upstaging: 11%-60%

Sensitive markers: serum, urine, imaging?

Tumorangiogenesis and PCaTumorangiogenesis and PCa

Microvessel density ~ Pathologic stageOrgan confined tumor vs. non organ confined tumor

n=31, retropubic Px, CD31, 200x, 0.74 mm2

Stage Mean RangeOrgan confined (n=23) 49.7 23-97Positive margins (n=5) 81.6 54-104N1 (n=3) 87.2 65-111

MVD in Prostatic CarcinomaMVD in Prostatic Carcinoma

CD31, 200x

Urine VEGF and T-Stage in PCaUrine VEGF and T-Stage in PCa

VEGF-Elisa (R&D Systems), TNM

n=14 (64.6, ± 7.5 yrs.)

T2: n=6, Urine VEGF = 191 pg/ml (± 75)

T3: n=8, Urine VEGF = 309 pg/ml (± 163)

p=0.04T2a/T2b and T3a/T3b: n.s.

Angiogenic Topography in PCaAngiogenic Topography in PCa

•PCa, rad Px, n=60 (64 ± 6 yrs)

•MVD endothelial antigen CD34

•VEGF165 monoclonal Ab

•7,9 mm2 area, serial sections

•Distribution and topography CD34+VEGF165

•Topography divided in 4 categories:

Identical, intersecting, adjacent, no contact

Angiogenic Topography in PCaAngiogenic Topography in PCa

CD34

VEGF

Angiogenic Topography in PCaAngiogenic Topography in PCa

•MVD between four groups n.s .•VEGF165-expression and highest MVD

-identical: 19 (31.6%) -intersecting: 18 (30%) -adjacent: 11 (18.3%) -no contact: 12 (20%)

•Close topographical relation in 80%

Angiogenesis and NEAngiogenesis and NE

•PCa with high NE differentiation: poor prognosis•NE and Neovascularisation?

•PCa, rad Px, n=102 (65.2 ± 6.6 yrs)

•Chromogranin A, CD34, 200x, morphometry

•NE: scattered cells

small clusters (<10 cells)

large clusters (>10 cells)

Angiogenesis and NEAngiogenesis and NE

Scattered cells14.6 ± 2.2

Small cluster82.8 ± 16.3

Large cluster291.3 ± 40.1

Chromogranin A, 200x

Angiogenesis and NEAngiogenesis and NE

•G Ia / Ib / IIa, Gleason 2-6, n=36

•G IIb / IIIa / IIIb, Gleason 7-10, n=66

•Low grade PCa: 33.8 ± 6.7 NE cells

•High grade PCa: 90.5 ± 16.4 NE cells

)p=0.028(

Angiogenesis and NEAngiogenesis and NE

1.33

±0.411,2

2.79

±0.163

2.56

±0.183MVD

201.8

±86.61,2

73.3

±15.23

49.6

±10.63NE tumor cells

pT4

n=6

pT3

n=58

pT2

n=36

11p<0.05 vs. pT2 tumors, p<0.05 vs. pT2 tumors, 22p<0.05 vs. pT3 tumors, p<0.05 vs. pT3 tumors, 33p<0.05 vs. pT4 tumorsp<0.05 vs. pT4 tumors

Angiogenesis and NEAngiogenesis and NE

•Low grade PCa, high NE

CD34: 2.4% ± 0.26

•High grade PCa, high NE

CD34: 3.3% ± 0.26

)p=0.026(

Angiogenesis, NEAngiogenesis, NEand Proliferationand Proliferation

•S-VEGF: high angiogenic potential

•Ki-67: proliferation

•Neuroendocrine differentiation (NE)

•Correlation of S-VEGF and Ki-67 in neuroendocrine differentiated PCa?

Angiogenesis, NE and Proliferation

•PCa, rad Px, T2b-3a, n=9, ( 65.8 yrs)

•S-VEGF (quantitative ELISA)

•Chromogranin A

•Ki-67 (MIB-1-Ab)

•Expression, topography

Angiogenesis, NE Angiogenesis, NE and Proliferationand Proliferation

•S-VEGF correlates with Ki-67 in areas of PCa with high NE differentiation .

)p=0.0228, r=0.67 (

•S-VEGF does not correlate with Ki-67 in areas with little NE differentiation.

)p=0.2110(

Vascular MorphogenesisVascular Morphogenesis

Microvessel MaturityMicrovessel MaturityNewly formed immature vessels

vs. Established and mature vessels

Coating of periendothelial cells:Pericytes, smooth muscle cells, myocardial cells

Anti-angiogenic therapy: MVD , change of ratio immature/mature vessels

Vulnerability of uncoated ECs

SMC-EC InteractionSMC-EC Interaction

•ECs initiate angiogenesis•Periendothelial cells for vascular maturation

Vascular myogenesis / mural cells

•Vessel stability•Inhibition of EC proliferation•Production of extracellular matrix•Viscoelastic and vasomotor properties•Specialized functions of ECs

AngioarchitectureAngioarchitecture

• HMW-MAA (MAb 225.28S)• Staining restricted to pericytes• Staining „clusters of cellular processes“

• Pericytes aquire HMW-MAA• HMW-MAA pos. pericytes = proliferation?• HMW-MAA in PCa?

Antibody supplied by S. Ferrone, Roswell Park Cancer Insitute, USA

Angioarchitecture HMW-MAAAngioarchitecture HMW-MAA

MAb 225.28S, 40x

Human Plasminogen - Kringle 5Human Plasminogen - Kringle 5

K1-4=Angiostatin™

K5

Kringle 5 in Urine Kringle 5 in Urine of Cancer Patientsof Cancer Patients

•Breast cancer

•Lung cancer

•Rheumatoid arthritis

•Prostate cancer

Band reacts only with K5 Ab, not K1-3 or K4 Abs

K5 has a molecular mass of 10459 kDa

HMVEC bFGF/VEGF Stimulated MigrationHMVEC bFGF/VEGF Stimulated Migration

Control (15 ng/ml bFGF/VEGF)

400 pM K5(15 ng/ml bFGF/VEGF)

40x magnification

36 Cells Migrated 2 Cells Migrated

0

20

40

60

80

100

120

Migration Inhibition of HMVEC Cells Migration Inhibition of HMVEC Cells by K5 with Various Activatorsby K5 with Various Activators

HGF/SFIL-8TGF-PDGFVEGFaFGFbFGFNone

En

doth

elia

l Cel

l Mig

rati

on

None

K5 (400 pM)

Pharmacokinetic MRI-ModelPharmacokinetic MRI-Model

k21 extracellular space

Gd

GdGd

Gd

GdGd

Am

pli

tud

e A

k21

Pharmacokinetic MRI-ModelPharmacokinetic MRI-Model

Amplitude A - Signal intensity - Vessel densityk21 - Exchangerate - Vessel density / Vessel permeability

Pharmacokinetic MRI-ModelPharmacokinetic MRI-Model

0

100

200

0 1,5 3 4,5 6 min

O2

0

100

200

0 1,5 3 4,5 6 min

Change of signal intensity [%]

MR-Spectroscopy in PCaMR-Spectroscopy in PCa

MR-Spectroscopy in PCaMR-Spectroscopy in PCa

PCa, before inhalation PCa, after inhalation

MR-Spectroscopy in PCaMR-Spectroscopy in PCa

Change of signal intensity in ROI

Angiogenic SurroundingsAngiogenic Surroundings•Tumorproliferation / Apoptosis•Microvessel sprouting•Recruitment of periendothelial cells•Angioarchitecture•Neuroendocrine differentiation•Hormonal influence (Androgens)

Paracrine pathway of angiogenic stimulation? Tumor microenvironment? Antiangiogenesis?

VEGF and Androgens in PCaVEGF and Androgens in PCa

Androgens induce VEGF in hormone-sensitive tissues

VEGF induces EC proliferation

Hormonal ablation in androgen-sensitive tumors

Withdrawal of tumor cell VEGF:Apoptosis of ECs AND tumor cells surrounding ECs

Regression of tumor mass

Problem: Recurrence due to hormone refractory variants

Antiangiogenic TrialsAntiangiogenic Trials

Natl Cancer Inst Database, 8/99Natl Cancer Inst Database, 8/99

Tumorangiogenesis and Tumorangiogenesis and Cancer:Cancer:

RelevanceRelevance??Diagnostic:

Screening (Serum, Urine)

Staging (Expression of GF, MVD, Imaging)

Prognosis

Therapeutic:

Antiangiogenesis (30 cancer drugs on trial NCI)

Market: $ 3 billion by 2005 (FT Pharmaceuticals)