Wykład I Mechanizmy angiogenezy

25.04.2010 Prof. dr hab. Józef Dulak

Zakład Biotechnologii Medycznej Wydział Biochemii, Biofizyki i Biotechnologii UJ

Email: jozef. dulak@uj.edu.pl

Web page: http://biotka.mol.uj.edu.pl/zbm

William Harvey (1578-1657) using a deer to demonstrate the circulatorysystem. His research laid the foundation for a scientific approach to medicine.

William Harvey - 1628 the first systematic description of circulatory system

chicken embryo

Marcello Malpighi – observed capillaries in 1661

Wilhelm His – 1865 – coined the term „endothelium”,to differentiate the inner lining of body cavities from „epithelium”

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 –description of lymphatic system –Henryk Hoyer – Jagiellonian University

Diagram of the organization of lymphatic and vein systems in chordate1, general schema; 2, lamprey; 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).

The vascular wall

Structure of blood vessels and lymphatics Structure of blood vessels and lymphatics

Jain R, Nature Med. June 2003

large vessel

Circulatory system and blood vessels

Endothelial cells

The crucial player in blood vessel formation

Endothelial cells in culture – cobblestone appearance

1973 & 1974 – Jaffe et al. & Gimbrone et al. –isolation of human endothelial cells (EC) from the umbilical vein

Jaffe EA, Nachman RL, Becker CG, Minick CRCulture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745-56

Gimbrone MA Jr , Cotran RS, Folkman J.Endothelial regeneration: studies with human endothelial cells in culture.Ser Haematol. 1973;6(4):453-5.Links

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. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolavers of closely opposed, polygonal large cellswhereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor'sblood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

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.

Endothelial cells

- one of the most quiescent and genetically stable cells of the body –turnover time is usually hundred of days

- proliferation is inhibited due to the contact with the capillary basement membrane

Endothelial cell markers used to identify microvasculature in tissues

Marker Ligand

CD31 (PECAM-1) CD31 on endothelial cells, leukocytes; glycosaminoglycans

CD34 L-selectin

CD54 (ICAM-1) LFA-1 integrin

CD62E (E-selectin) Sialyl-Lewis-X antigen and other carbohydrates

CD105 (endoglin) TGF-β1 and –β3

CD106 (VCAM-1) VLA-4 integrin

CD141 (thrombomodulin) Thrombin

CD144 (VE-cadherin) CD144 homotypic interaction

Endothelin receptor B (ETBR) ET-1

Ephrin B2 EphB4

EphB4 Ephrin B2

Tie-2 Angiopoietins

VEGFR-2 VEGF

von Willebrand Factor (vWF) Factor VIII

Uptake of Ac-LDL Scavenger receptor

Morphological differentiation of endothelial cells Morphological differentiation of endothelial cells

Skeletal muscle, heart,lung, brain

Endocrine and exocrine organsintestinal villi

Cleaver O & Melton DA, Nature Med., June 2003

How the blood vessels are formed?

Blood vessel formationa.) 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

Blood vessel formation

• Vasculogenesis: a.) during embryonic development;

• Angiogenesis:a.) embryonic development

b.) adulthood: wound healing, menstrual cycle, tumour-angiogenesis…

b.) during adulthood associated with circulating progenitor cells

angioblast capillary

bFGF

VEGF

VEGF

Ang1, bFGF

Vasculogenesiscapillaries are formed from

vascular progenitor cells

Angiogenesis formation of new blood vessels from pre-existing vessels

Arteriogenesisformation of mature blood vessels; differentiation into

veins and arteries

Three ways of formation of blood vessels Three ways of formation of blood vessels

MCP-1, PDGF

Ang-2

Major growth factors and receptors involvedin blood vessels formation

VEGF – vascular endothelial growth factorsVEGF-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 – fibroblasts growth factors

PDGF – platelet-derived growth factor

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

Model organisms in vascular research

Vasculogenesis Vasculogenesis begins very early after the initiation of gastrulation in the mammalian embryo, with the formation of the blood islands in the yolk sac and angioblast precursors in the head mesenchyme

The human yolk sac is a membrane outside the embryo that is connected by a tube (the yolk stalk) though the umbilical opening to the embryo's midgut. The yolk sac serves as an early site for the formation of blood and in time is incorporated into the primitive gut of the embryo

capillaries are formed from vascular progenitor cells

Vasculogenesis

1. First phase• Initiated from the generation of hemangioblasts;

2. Second phase• Angioblastsproliferate and differentiate into

endothelial cells

3. Third phase

• Endothelial cells form primary capillary plexus

Formation of a vascular network

Hematopoiesis in zebrafish (Danio rerio)

Expression of the flk-1 represents the earliest marker of the developing endothelial lineage during vasculogenesis

SCL transcription factor is crucial for the developmentof blood cells and blood vessels

Common vascular progenitor cells Common vascular progenitor cells for endothelial cells and for endothelial cells and

vascular smooth muscle cells vascular smooth muscle cells

Hemangioblast

Angioblast

Venous Arterialendothelium endothelium

VEGF

Commonvascular

progenitor cells

(flk-1+)

VEGF

PDGF-BBPericytes/smooth muscles

progenitor cells of vascular smooth muscles

(various forms)

Blood cells Skeletal muscles

Origin of endothelial and mural cells from different progenitor cells

PDGF-BB

Vasculogenesis in adult

Vasculogenesis occurs also in adult organismVasculogenesis occurs also in adult organism

Previously, postnatal vascularization was thought to occur exclusively due to angiogenesis. However, recent investigations have shown that vasculogenesis is involved in blood vessel formation also during postnatal live and the cells responsible for that are EPCs.

JCI 1999;103:1231-1236

• Classic Paradigm: Angiogenesis– Mature ECs migrate and

proliferate to form new vessels

• New Model:Angiogenesis + Vasculogenesis– Bone Marrow derived EPCs

circulate to sites of neovascularization

Progenitor cells

Ancestor cells that can form mature cells to restore function in tissues

Endothelial progenitor cells

A primitive cell made in the bone marrow (or otherorgans) that can enter the bloodstream and go to areas

of blood-vessel injury to help repair the damage

Stem cells

Undifferentiated cells that can develop into any type of cell in the body

Adult stem cellsAdult stem cells

hematopoietic stem cells

mesenchymal stem cells

bone marrow

13

Differentiation patwhays for pluripotent bone marrow stromal cells

Plasticity of adult stem cells

the ability to form specialized cell types of other tissues

(also called transdifferentiation)

14

Endothelial Progenitor Cells

• 1997 Asahara et al. reported putative EPCs or angioblasts

– Cells display progenitor phenotype

– Differentiate into mature ECs in vitro– Participate in neovascularization when injected

into ischemic animal model

Science 1997;275:964-967

DiI AcLDL uptake

EPC

1:200 2h

Isolectin binding by EPC

control

isolectin

Lectin from Bandeira simplicifolia

Immunohistochemical characterisation of culture-expanded EPCs

Cytoplasmic factor VIII (von Willebrand factor)

Uptake of acetylated low density lipoproteins

2 weeks culture

tube formation on Matrigel

Dzau et al., Hypertension 2005,

Markers of endothelial progenitor cells

CD34VEGFR2CD133 (prominin, AC133)

CD133 is absent on mature endothelial cells and monocytic cells

Numerous origins of endothelial progenitor cells Numerous origins of endothelial progenitor cells

Urbich & Dimmeler, Trends Cardiovasc Med. 2004

Mechanisms of EPC homing and differentiation

Urbich & Dimmeler, Circ Res 2004

Vascular progenitors in the embryo and in the adults

Blood vessel formationa.) 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

Angiogenesis

• Majority of vascular development occurs via angiogenesis

• Growth of new blood vessels from existing vessels

• Two distinct mechanisms available

a.) sprouting angiogenesisb.) intussusceptive angiogenesis

Sprouting angiogenesis

• Sprouting: invasion of new capillaries into unvascularized tissue from existing mature vasculature

- degradation of matrix proteins

- detachment and migration of ECs

- proliferation

Sprouting angiogenesis

Intussusceptiveor non sprouting angiogenesis

- remodelling of existing vessels

- interendothelial contact is needed

- splits into two vessels

a: Remodeling: A pillar appears in close proximity of the branching angle

b,c: From the pillar a fold develops toward the tissue of the branching angle and finally the pillar tissue merges with the interstitium of the branching angle

d: As a result, the branching angle is relocated proximalad and the angle is altered

e: Pruning: A column of pillars arises close to the branching angleThe pillars can merge along a line indicated by the arrows. The eccentric location of the pillars can lead to alterations in the diameter of a vascular branch.

f: Ultimately, a branch can be completely pruned 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

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Etapy angiogenezy Etapy angiogenezy

� zwiększenie przepuszczalności ściany i odkładanie włóknika�rozluźnienie warstwy komórek wspomagających (pericytów) � uwolnienie proteinaz przez komórki śródbłonka � trawienie błony podstawnej i macierzy pozakomórkowej otaczającej

naczynie krwionośne�migracja i proliferacja komórek śródbłonka� tworzenie struktur naczyniowych � łączenie (fuzja) nowych naczyń� inicjacja przepływu krwi

- zahamowanie proliferacji komórek śródbłonka - zahamowanie migracji komórek śródbłonka

� synteza błony podstawnej

Stages of angiogenesis Stages of angiogenesis

� increase in vessel permeability and thrombin deposition � loosening of pericyte contact � proteinase release from endothelial cells � digestion of basement membrane and extracellular matrix �migration and proliferation of endothelial cells � formation of vascular structures � fusion of new vessels � initiation of blood flow

- inhibition of endothelial cell proliferation - inhibition of the migration of endothelial cells

� formation of basement membrane

Endoglin is an auxiliary receptor for the transforming growth factor-beta family of cytokines and is required for angiogenesis and heart development.

Summary of the mechanisms of angiogenesis

arterio/venous

differentiation(ephrins/Eph)

Błona podstawna(basement membrane)

Specialised form of extracellular matrix

Basement membrane in various organs

Basement membrane of the blood vessels

- collagen IV - collagen XV- collagen XVIII- laminin - heparan-sulphate proteoglycans- perlecans - nidogen/entactin - SPARC/BM-40/osteopontin

Crucial role of metalloproteinases in angiogenesis

MMP-2 – gelatinase AMMP-9 – gelatinase B

MMP family MMP Name ECM Substrate

Collagenases Interstitial Collagenase (MMP-1) fibrillar collagens

Neutrophil Collagenase (MMP-8)

Collagenase-3 (MMP-13)

MMP-18 (Collagenase 4, xenopus collagenase)

Stromelysins Stromelysin-1 (MMP-3) laminin, fibronectin, non-fibrillar collagens

Stromelysin-2 (MMP-10)

Stromelysin-3 (MMP-11)

Matrilysins Matrilysin (MMP-7) PUMP laminin, fibronectin, non-fibrillar collagens

MMP-26 (Matrilysin-2, endometase)

The membrane-bound MMPs

MT1-MMP (MMP-14) gelatinase A, fibrillarcollagens, proteoglycans,ECM glycoproteins

MT2-MMP (MMP-15) gelatinase A

MT3-MMP (MMP-16) gelatinase A

MT4-MMP (MMP-17)

Gelatinases Gelatinase A (MMP-2) type I, IV, V and fibrillarcollagens; gelatin

Gelatinase B (MMP-9) type IV, V collagen,gelatin

Others MMP (examples) MMP-19 (RASI-1) gelatin

Enamelysin (MMP-20) type V collagen

metalloelastase (MMP-12) elastin

Metalloproteinases Metalloproteinases

Prodomain Catalytic domain Hemopexin

PRCGxPD

gelatinbinding

Zn2+

Zn2+

Zn2+

Zn2+

Zn2+

Collagenase family

Gelatinase family

MT-MMPs

MMP-7, MMP-23,MMP-26

Hingeregion

Stromelysin family

Transmembrane

Matrix metalloproteinases

Tissue inhibitors of metalloproteinases (TIMPs)

Proteolytic enzymes

Serine proteinase (plasminogen activators)

Matrix metalloproteinases (MMPs)zinc-dependent endopeptidases

Differentiation of endothelial cells on basement membrane Differentiation of endothelial cells on basement membrane

Physiological and pathological angiogenesis Physiological and pathological angiogenesis

Physiological angiogenesis in adults is restricted Physiological angiogenesis in adults is restricted

placenta uterus

Hair growthWound healing

New capillary formation in response to wounding

RheumathoidRheumathoidArthritis Arthritis

TumorsTumorsAIDS AIDS

complicationscomplications

Psoriasis Psoriasis

Infertility Infertility

SclerodermiaSclerodermia

Cardiovascular Cardiovascular diseasesdiseases

UlcersUlcers

StrokeStroke

Sight lossSight loss

angiogenesis

Exagerrated Exagerrated

InsufficientInsufficient

Take home messages

1. Three mechanisms of formation of blood vessels

2. Common origin of blood cells and endothelial cells

3. Endothelial progenitor cells play a role in blood vessel formation also in adults

4. Numerous mediators are involved in blood vessels formation

5. Physiological angiogenesis in adults is restricted, but it is a significant component of numerous diseases, such as cancer or atherosclerosis