PhD program N090/N094

Vascular BiologyJune 2008

Vasculogenesis, angiogenesis and lymphangiogenesis:Signaling pathways

Erhard Hofer

Department of Vascular Biology and Thrombosis ResearchCenter for Biomolecular Medicine and PharmacologyMedical University of Vienna

Vasculogenesis, angiogenesis and lymphangiogenesis:Signaling pathways

Vasculogenesis - angiogenesis - arteriogenesisvasculogenesis - developmentsprouting angiogenesisarteriogenesislymphangiogenesis

Important factors and receptorslessons from ko micehypoxia - HIF - VEGFEC-specific ligands + receptorsdownstream signals - genes - proteins

Angiogenesis and diseasestumor angiogenesisother diseases with excess or deficiency in angiogenesis

Literature:

Books:

B. Alberts et al., Molecular Biology of the Cell,5th Edition, Taylor and Francis Inc., 2007Pg. 1279-1283

Endothelial BiomedicineW. Aird, ed.Cambridge University Press, 2007

R.A. Weinberg, The Biology of Cancer, Garland Science, 2007Pg. 556-585

Tumor Angiogenesis - Basic mechanisms and Cancer Therapy, D. Marme, N. Fusenig, ed.Springer Verlag 2008

Nature Insight Angiogenesis G.D. Yancopoulos et al. (2000). Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248.

Angiogenesis Focus, Nature Med 9, June 2003Peter Carmeliet, Angiogenesis in Health and DiseaseNapoleone Ferrara et al., The biology of VEGF and its receptorsRakesh K. Jain, Molecular regulation of vessel maturationShanin Rafii and David Lyden, Therapeutic stem and progenitor celltransplantation for organ vascularization and regenerationChristopher W. Pugh and Peter J. Ratcliffe, Regulation of angiogenesis by hypoxia: role of the HIF system

Angiogenesis, Nature Reviews Cancer 3, June 2003 Gabriele Bergers and Laura E. Benjamin, Tumorigenesis and the angiogenic switch

Literature:

Reviews:

C.J. Schofield and P.J. Ratcliffe. Oxygen sensing by HIF hydroxylases.Nature Rev. Mol. Cell Biol. 5, 343-354 (2004)

Nature Insight Angiogenesis, Vol. 438, pg. 931-974, December 2005Carmeliet, Angiogenesis in life, disease and medicine Coultas, Endothelial cells and VEGF in vascular development Alitalo, Lymphangiogenesis in development and human disease Greenberg, From angiogenesis to neuropathology Gariano, Retinal angiogenesis in development and disease Ferrara, Angiogenesis as a therapeutic target

P. Carmeliet and M. Tessier-Lavigne, Common mechanisms of nerve and blood vessel wiring, Nature 436, 195-200 (2005)

J. Folkman, Angiogenesis: an organizing principle for drug discovery ?Nature Reviews Drug Discovery 6, 273-286 (2007)

S. Zacchigna et al., Similarities between angiogenesis and neural development: what small animal models can tell us. Curr.Top.Dev.Biol. 80, 1-55 (2008)

Stem cells and vascular progenitor cells:

Angiogenesis Focus, Nature Med 9, June 2003Shanin Rafii and David Lyden, Therapeutic stem and progenitor cell

Nature Insight Angiogenesis, Vol. 438, pg. 931-974, December 2005L. Coultas et al., Endothelial cells and VEGF in vascular development

Nature Insight Cardiovascular Disease, Vol. 451, 903, February 2008V.F.M. Segers and R.T. Lee, Stem-cell therapy for cardiac disease

Nature Insight Regenerative Medicine, Vol. 453, 7193, May 2008 R. Passier et al., Stem-cell-based therapy and lessons from the heart

H. Bai and Z.Z.Wang, Directing human embryonic stem cells toGenerate vascular progenitor cells. Gene Therapy 15, 89-95, 2008.

L. Yang et al., Human cardiovascular progenitor cells develop fromA KDR+ embryonic-stem-cell-derived population. Nature 453, 524-528, May 2008

D. Gao et al., Endothelial progenitor cells control the angiogenic switchIn mouse lung metastasis. Science 319, 195-198, 2008.

Unterlagen:

http://mailbox.univie.ac.at/erhard.hoferStudent point, Vorlesungsunterlagen

erhard.hofer@meduniwien.ac.at

1- Vasculogenesis - Angiogenesis - Arteriogenesis

VasculogenesisFormation of blood vessels by differentiation from (hem)angioblasts

Sprouting angiogenesisSprouting of cells from mature endothelial cells of the vessel wall

Arteriogenesisgrowth of large arteries from pre-existing small vessels/capillaries

LymphangiogenesisFormation of the lymphatic vasculature

Vasculogenesis

Development from mesoderm

Connective tissueBlood cellsBlood vesselsMuscle

Etc.

Vasculogenesis

Formation of vessels by differentiation of cells from angioblasts in the yolk sac of the embryo:

Is differentiation and proliferation of endothelial cells in a non-vascularized tissue

Leads to formation of a primitive tubular network

Has to undergo angiogenic remodeling to stable vascular system

Gene mutations in mice

HIF1a -/- embryonic lethal E11

VEGFR1 -/- embryonic lethal E8.5-9.5, overgrowth of endothelial vasculatureVEGFR1 TK del -/- healthyVEGFR2 -/- embryonic lethal E8.5, impaired hematopoietic and ECVEGFR3 -/- 90% lethal soon after E9.5, reduced and disorganized EC

VEGF-A +/- heterozygous lethal E9.5, impaired vasculogenesisVEGF-B -/- viable with smaller heart, dysfunctional coronary vasculatureVEGF-C -/- embryonic lethal E15.5, failure in lymphangiogenesis

Notch1 -/- embryonic lethal E11, failure of vascular remodelingJag1 -/- embryonic lethal E9.5-11.5, failure of vascular remodeling

Tie-1 -/- embryonic lethal E13.5, loss of vascular integrity Tie-2 -/- embryonic lethal E9.5

Ang-1 -/- phenocopies Tie-2Ang-2 -/- viable, but vascular defects

Hemangioblast Angioblast EC

Postnatal vasculogenesis

Structure of vessels and capillaries

Monocellular layer of endothelial cellsSmall artery:

Capillary: endothelial cell, basal lamina, pericytes

Life time of endothelial cells:

months (lung, liver) to years (brain, muscle)

Slow repair and renewal of vascular wall

New vessel formation:

Embryo, growthIn uterus, during menstruation cycleWound repair

Mouse cornea:wounding induces angiogenesis,chemotactic response toangiogenic factors

Angiogenesis:Sprouting of cells from mature endothelial cells of the vessel wall

secretion of proteases, resolution ofbasal lamina, migration towards chemotactic gradient, proliferation,tube formation

VEGF is factor largely specific for endothelial cells,bFGF can also induce, not specific for EC

tip cell

stem

capillaries sprouting in the retina of an embryonic mouse

capillary lumen opening up

behind the tip cell(red dye injected)

Sprouting towards chemotactic gradient: VEGF

Hypoxia - HIF - VEGFevery cell must be within 50 to 100 m of a capillary

HIF: hypoxia inducible factorVEGF: vascular endothelial growth factor

VEGF-gene:Regulated by HIF,HIF is continously produced,ubiquitinylated, degraded in proteasome,therefore low concentration;

Ubiquitinylation dependent onHippel-Lindau tumor suppressor(part of an E3 ubiquitin-ligase complex)

HIF1is modified by a prolyl hydroxylase,then better interaction with vHL protein, high turnover;Hydroxylase is regulated by O2

Von Hippel-Lindau Tumor Suppressor, HIF and VEGF

FIH: factor inhibiting HIFHIF asparginyl hydroxylase

Factors and receptors

Endothelium-specific factors:VEGF family: 5 factorsAngiopoietin family : 4 factorsEphrin family : at least 1 factor

Non EC-specific factors :bFGFPDGFTGF-

VEGF/VEGFR:VEGF-A: initiation of vasculogenesisand sprouting angiogenesis,Immature vessels,Vascular permeability factor,Haploid insufficiency in k.o. mice,

PlGF: remodeling of adult vessels VEGF-B: heart vascularization ?VEGF-C: lymphatic vesselsVEGF-D: lymphatic vessels ?

VEGFR-2: growth and permeabilityVEGFR-1: negative role ?, decoy receptor,

synergism with VEGFR-2 in tumor angiogenesis VEGFR-3: lymphatic vessels

VEGF/VEGFR family

3 important Signaling cascades can be induced

- Ras

- PLC-(Phospholipase C-

- PI3-Kinase(Phosphoinositol 3-Kinase)

Docking of proteins via SH2 (Src-homology) Domainsbinds P-Tyr and neighbouring amino acidsWas originally described for the intracellular Tyr-Kinase c-Src (Onkogen of Rous Sarcoma Virus)

Grb-2 Adaptor: SH2- Domain

SOS is a Ras-GEF (guanine nucleotide exchange factor)

Ras: GTP-binding Protein (Onkogen detected in Rat Sarcoma)

SOS

Ras activates MAP-KinasePathway

1- MAPKKK2- MAPKK3- MAPK

MAPK: Mitogen-activated Kinase

(there are three MAP-Kinase cascades:MEK/ERKP38JNK)

Raf

MEK

ERK

The phosphorylated MAPK ERK is transported into the nucleusand phosphorylates the transcription factor TCF

ERK: extracellular signal regulated kinaseTCF: ternary complex factorSRF: serum response factorSRE: serum response element

(DNA binding sequence for TCF and SRF in promoter of different genes)

Genes forCell cycle/Proliferation,e.g. c-Fos

PKB, PDK:(PDK: PI-dependent kinase)

Ser/Thr kinases

PI-3 Kinase Pathway and Survival

aktivierte PLC-

PKCphosphoryliert viele Substrate,kann MAP Kinase Signalweg induzieren, Genregulation

Ca++

Calmodulin/CalcineurinNFAT- Trankriptionsfaktor

PLC- signaling pathway

10-7 M

10-3 M

„Second messenger“DAG, IP3 and Ca++

Ca++

Calmodulin

Calcineurin

NFAT

PI

Ca++ signaling pathway/gene regulation

the phosphatasecalcineurindephosphorylatesNFAT

NFAT translocatesInto the nucleus

NFAT= transcription factor(nuclear factoractivated T cell)

nucleus

P

Differential signaling by tyr kinase receptors

gene regulation

proliferationvasculogenesisangiogenesis

Y799

Y820

Y925

Y936

Y951Y994

Y1006

Y1052Y1057

Y1080Y1104

Y1128Y1134

Y1175Y1212Y1221

Y1303Y1307

Y1317

P38, src (vascular leakage?)

TSAd (migration)

PI-3 kinase (survival)

PLC-

EC “specific” factors/receptors:

VEGFR1 VEGF-A, PLGF

VEGFR2 VEGF-AVEGFR3 VEGF-CTIE1 ?TIE2 ANG1,2

VEGFR2

Sakurai et al.PNAS 2005

VEGF-AVEGFR2 PLC-

Ca++ PKC/calcineurin/ MAPK NFAT EGR-1

cooperative gene regulation

NAB2

CsADSCR-1

EGFHER1Ras

EGR-1

gene regulation

VEGFR2 vs. EGFR signaling

other signals

other signals MAPK

Guidance molecules in endothelial tip cell attraction and repulsion

Eichmann A, Curr Opin Neurobiol. 2005

Carmeliet P, Nature. 2005

Angiopoietins und Tie Receptors:

Ang1: remodeling and maturationQuiescence and stabilityResistance to permeability,Supports interaction with other cells and matrix,Vessel size (VEGF number of vessels),Repair of damaged vessels

Ang2: natural antagonist,Overexpression similar Ang-1 k.o. oder Tie-2 k.o.,Destabilization signal for initiation of vascular remodelingEither regression or increased VEGF sensitivityAng2 is induced in tumors

Ang3: ?Ang4: ?

Tie2: binds Ang1-4

Tie1: ?

Ephrine und Eph-Rezeptors:

Largest family of growth factor receptors,Relevant for vascular system:Ephrin B2/ Eph B4 : remodeling and maturation Different for early arterial (Ephrin B2) and venous vessels (EphB4),Hypothesis: role for fusion of arterial/venous vessels

Arteriogenesis

Growth of arteries from pre-existing small capillaries

MacrophagesMCP-1

Figure 13.31 The Biology of Cancer (© Garland Science 2007)

Network of lymphatic vessels (red) and capillaries (green):Lymphatic vessels are larger, not supported by underlying mural cells

1-Sprouting

3-incorporation of BM-derived precursors

5-Lymphangiogenesis

Growth of tumor vessels

2-Intussusceptive growth

4-Cooption of existing vessels

Rolle von VEGF und Ang2 bei der Tumorangiogenese,VEGF-Blockade vielversprechende Anti-Angiogenese Therapie

Concept 2: many tumors “home in” onto vessels, occupate existing vessels,Vessel produces Ang2, first tumor regression, then VEGF production by tumor

Concept 1: non-vascularized Tumor

Figure 13.32a The Biology of Cancer (© Garland Science 2007)

Recruitment of capillaries by an implanted tumor

Figure 13.34a The Biology of Cancer (© Garland Science 2007)

Chaotic organization of tumor-associated vasculature

Structure and function of tumor vessels:

Chaotic architecture and blood flowTherefore hypoxic and acidic regions in tumorPermeability strongly increased

Fenestraeenlarged Junctions

No functional lymphatics inside the tumorenlarged in surrounding,increases metastasis

Mosaic vessels

Abnormales Endothelium

Figure 13.33 The Biology of Cancer (© Garland Science 2007)

Tumor vessel is only partially overlaid by pericytes and SMC

Figure 13.37 The Biology of Cancer (© Garland Science 2007)

The Rip-Tag model of islet tumor cell progressionTransgene: SV40 large and small T transcription driven by insulin promoter

Transcription in b-cells of islets of Langerhans

Figure 13.38b The Biology of Cancer (© Garland Science 2007)

The angiogenic switch and recruitment of inflammatory cells

Angiogenesis-dependent diseases

excessCancer

Infantile hemangiomas

Autoimmune diseases, chronic inflammatory diseases:Rheumatoid arthritisPsoriasis

Age-related macular degeneration

Atherosclerosis

Deficiency:Limb ischemiaMyocardial ischemia